CA6 antigen-specific cytotoxic conjugate and methods of using the same

ABSTRACT

Cytotoxic conjugates comprising a cell binding agent and a cytotoxic agent, therapeutic compositions comprising the conjugate, methods for using the conjugates in the inhibition of cell growth and the treatment of disease, and a kit comprising the cytotoxic conjugate are disclosed are all embodiments of the invention. In particular, the cell binding agent is a monoclonal antibody, and epitope-binding fragments thereof, that recognizes and binds the CA6 glycotope. The present invention is also directed to humanized or resurfaced versions of DS6, an anti-CA6 murine monoclonal antibody, and epitope-binding fragments thereof.

RELATED APPLICATIONS

The present application is a continuation-in-part application of priorpending application Ser. No. 10/895,135, filed Jul. 21, 2004, whichclaims benefit of U.S. Provisional Application No. 60/488,447, filedJul. 21, 2003. Both applications are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention is directed to a murine anti-CA6 glycotopemonoclonal antibody, and humanized or resurfaced versions thereof. Thepresent invention is also directed to epitope-binding fragments of theanti-CA6 glycotope monoclonal antibody, as well as to epitope-bindingfragments of humanized or resurfaced versions of the anti-CA6 glycotopemonoclonal antibody.

The present invention is further directed to cytotoxic conjugatescomprising a cell binding agent and a cytotoxic agent, therapeuticcompositions comprising the conjugate, methods for using the conjugatesin the inhibition of cell growth and the treatment of disease, and a kitcomprising the cytotoxic conjugate. In particular, the cell bindingagent is a monoclonal antibody, or epitope-binding fragment thereof,that recognizes and binds the CA6 glycotope or a humanized or resurfacedversion thereof.

BACKGROUND OF THE INVENTION

There have been numerous attempts to develop anti-cancer therapeuticagents that specifically destroy target cancer cells without harmingsurrounding, non-cancerous cells and tissue. Such therapeutic agentshave the potential to vastly improve the treatment of cancer in humanpatients.

One promising approach has been to link cell binding agents, such asmonoclonal antibodies, with cytotoxic drugs (Sela et al, inImmunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al, in TargetedDrugs 1-22 (E. Goldberg, ed. 1983); Diener et al, in Antibody mediateddelivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et al, inAntibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988); Bumolet al, in Antibody mediated delivery systems 55-79 (J. Rodwell, ed.1988). Depending on the selection of the cell binding agent, thesecytotoxic conjugates can be designed to recognize and bind only specifictypes of cancerous cells, based on the expression profile of moleculesexpressed on the surface of such cells.

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil havebeen used in such cytotoxic conjugates, linked to a variety of murinemonoclonal antibodies. In some cases, the drug molecules were linked tothe antibody molecules through an intermediary carrier molecule such asserum albumin (Garnett et al, 46 Cancer Res. 2407-2412 (1986); Ohkawa etal 23 Cancer Immunol. Immunother. 81-86 (1986); Endo et al, 47 CancerRes. 1076-1080 (1980)), dextran (Hurwitz et al, 2 Appl. Biochem. 25-35(1980); Manabi et al, 34 Biochem. Pharmacol. 289-291 (1985); Dillman etal, 46 Cancer Res. 4886-4891 (1986); Shoval et al, 85 Proc. Natl. Acad.Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al, 73 J. Natl.Canc. Inst. 721-729 (1984); Kato et al 27 J. Med. Chem. 1602-1607(1984); Tsukada et al, 52 Br. J. Cancer 111-116 (1985)).

As an example of one specific conjugate that has shown some promise, isthe conjugate of the C242 antibody, directed against CanAg, an antigenexpressed on colorectal and pancreatic tumors, and the maytansinederivative DM1 (Liu et al., Proc Natl Acad Sci USA, 93: 8618-8623(1996)). In vitro evaluation of this conjugate indicated that itsbinding affinity towards CanAg expressed on the cell surface was highwith an apparent K_(d) value of 3×10⁻¹¹ M, and its cytotoxic potency forCanAg-positive cells was high with an IC₅₀ of 6×10⁻¹¹ M. Thiscytotoxicity was antigen-dependent since it was blocked by an excess ofnon-conjugated antibody, and since antigen-negative cells were more than100-fold less sensitive to the conjugate. Other examples of antibody-DM1conjugates with both high affinity towards respective target cells andhigh antigen-selective cytotoxicity include those of huN901, a humanizedversion of antibody against human CD56; huMy9-6, a humanized version ofantibody against human CD33; huC242, a humanized version of antibodyagainst the CanAg Muc1 epitope; huJ591, a deimmunized antibody againstPSMA; trastuzumab, a humanized antibody against Her2/neu; andbivatuzumab, a humanized antibody against CD44v6.

The development of additional cytotoxic conjugates that specificallyrecognize particular types of cancerous cells will be important in thecontinuing improvement of methods used to treat patients with cancer.

To that end, the present invention is directed to the development ofantibodies that recognize and bind molecules/receptors expressed on thesurface of cancerous cells, and to the development of novel cytotoxicconjugates comprising cell binding agents, such as antibodies, andcytotoxic agents that specifically target the molecules/receptorsexpressed on the surface of cancerous cells.

More specifically, the present invention is directed to thecharacterization of a novel CA6 sialoglycotope on the Muc1 mucinreceptor expressed by cancerous cells, and to the provision ofantibodies, preferably humanized antibodies, that recognize the novelCA6 sialoglycotope of the Muc1 mucin and that may be used to inhibit thegrowth of a cell expressing the CA6 glycotope in the context of acytotoxic agent.

SUMMARY OF THE INVENTION

The present invention includes antibodies that specifically recognizeand bind a novel CA6 sialoglycotope of the Muc1 mucin receptor, or anepitope-binding fragment thereof. In another embodiment, the presentinvention includes a humanized antibody, or an epitope-binding fragmentthereof, that recognizes the novel CA6 sialoglycotope (“the CA6glycotope”) of the Muc1 mucin receptor.

In preferred embodiments, the present invention includes the murineanti-CA6 monoclonal antibody DS6 (“the DS6 antibody”), and resurfaced orhumanized versions of the DS6 antibody wherein surface-exposed residuesof the antibody, or its epitope-binding fragments, are replaced in bothlight and heavy chains to more closely resemble known human antibodysurfaces. The humanized antibodies and epitope-binding fragments thereofof the present invention have improved properties in that they are muchless immunogenic (or completely non-immunogenic) in human subjects towhich they are administered than fully murine versions. Thus, thehumanized DS6 antibodies and epitope-binding fragments thereof of thepresent invention specifically recognize a novel sialoglycotope on theMuc1 mucin receptor, i.e., the CA6 glycotope, while not beingimmunogenic to a human. The humanized antibodies and epitope-bindingfragments thereof can be conjugated to a drug, such as a maytansinoid,to form a prodrug having specific cytotoxicity towardsantigen-expressing cells by targeting the drug to the Muc1 CA6sialoglycotope. Cytotoxic conjugates comprising such antibodies andsmall, highly toxic drugs (e.g., maytansinoids, taxanes, and CC-1065analogs) can thus be used as a therapeutic for treatment of tumors, suchas breast and ovarian tumors.

The humanized versions of the DS6 antibody of the present invention arefully characterized herein with respect to their respective amino acidsequences of both light and heavy chain variable regions, the DNAsequences of the genes for the light and heavy chain variable regions,the identification of the CDRs, the identification of their surfaceamino acids, and disclosure of a means for their expression inrecombinant form.

In one embodiment, there is provided a humanized DS6 antibody or anepitope-binding fragment thereof having a heavy chain including CDRshaving amino acid sequences represented by SEQ ID NOS:1-3: (SEQ IDNO: 1) S Y N M H, (SEQ ID NO: 2) Y I Y P G N G A T N Y N Q K F K G, (SEQID NO: 3) G D S V P F A Y,

and having a light chain that comprises CDRs having amino acid sequencesrepresented by SEQ ID NOS:4-6: (SEQ ID NO: 4) S A H S S V S F M H, (SEQID NO: 5) S T S S L A S, (SEQ ID NO: 6) Q Q R S S F P L T,

Also provided are humanized DS6 antibodies and epitope-binding fragmentsthereof having a light chain variable region that has an amino acidsequence that shares at least 90% sequence identity with an amino acidsequence represented by SEQ ID NO:7 or SEQ ID NO: 8: (SEQ ID NO: 7)QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAG TKLELKR (SEQ ID NO:8) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAG TKLELKR

Similarly, there are provided humanized DS6 antibodies andepitope-binding fragments thereof having a heavy chain variable regionthat has an amino acid sequence that shares at least 90% sequenceidentity with an amino acid sequence represented by SEQ ID NO:9, SEQ IDNO:10, or SEQ ID NO: 11: (SEQ ID NO: 9)QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKEKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA(SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGDS VPFAYWGQGTLVTVSA (SEQID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA

In another embodiment, humanized DS6 antibodies and epitope-bindingfragments thereof are provided having a humanized or resurfaced lightchain variable region having an amino acid sequence corresponding to SEQID NO: 8 (SEQ ID NO: 8)EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWTYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAG TKLELKR.

Similarly, humanized DS6 antibodies and epitope-binding fragmentsthereof are provided having a humanized or resurfaced heavy chainvariable region having an amino acid sequence corresponding to SEQ IDNO:10 or SEQ ID NO: 11, respectively: (SEQ ID NO: 10)QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.(SEQ ID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.

The humanized DS6 antibodies and epitope-binding fragments thereof ofthe present invention can also include substitution in light and/orheavy chain amino acid residues at one or more positions defined by thestarred residues in Table 1 which represent the murine surface frameworkresidues found within 5 Angstroms of a CDR requiring change to a humanresidue. For example, the first amino acid residue Q in the murinesequence (SEQ ID NO:7) has been replaced by E (SEQ ID NO:8) to humanizethe antibody. However, because of the proximity of this residue to aCDR, a back mutation to the murine residue Q may be required to maintainantibody affinity. TABLE 1 muDS6 framework residues proximal to a CDR(Kabat numbering) Light chain Heavy chain Q1* Q1 V3 K64* T5 P73* P40 S74G57 A60 S67 E81

This is further shown in Table 2 where muDS6 variable region surfaceresidues are shown aligned with the three most homologous human variableregion surface residues. The amino acid residues in Table 1 correspondto the underlined amino acid residues in Table 2. TABLE 2 Top 3 MostHomologous Human Antibody Surfaces Antibody Light Chain SEQ ID NO: muDS6Q V T A I P K P G G A S R E K SEQ ID NO: 12 28E4 E V T A T P R P G G A SS E K SEQ ID NO: 13 HAZcPB E V T G T P R P G G D S R E K SEQ ID NO: 14SSaPB E V T G T P R P G G D S R E K SEQ ID NO: 15 Antibody Heavy ChainSEQ ID NO: muDS6 Q Y Q A L R S K K P G Q Q K K G P S S S E Q S SEQ IDNO: 16 28E4 Q Q V A V K P K K P G Q Q K Q G T S S S E Q S SEQ ID NO: 17HAZcPB — Q V A V K P K K P G Q Q K Q G E S S S E Q S SEQ ID NO: 18 SSaPB— Q V A V K P K K P G Q Q K Q G E S S S E Q S SEQ ID NO: 19

The present invention further provides cytotoxic conjugates comprising(1) a cell binding agent that recognizes and binds the CA6 glycotope,and (2) a cytotoxic agent. In the cytotoxic conjugates, the cell bindingagent has a high affinity for the CA6 glycotope and the cytotoxic agenthas a high degree of cytotoxicity for cells expressing the CA6glycotope, such that the cytotoxic conjugates of the present inventionform effective killing agents.

In a preferred embodiment, the cell binding agent is an anti-CA6antibody or an epitope-binding fragment thereof, more preferably ahumanized anti-CA6 antibody or an epitope-binding fragment thereof,wherein a cytotoxic agent is covalently attached, directly or via acleavable or non-cleavable linker, to the antibody or epitope-bindingfragment thereof. In more preferred embodiments, the cell binding agentis the humanized DS6 antibody or an epitope-binding fragment thereof,and the cytotoxic agent is a taxol, a maytansinoid, CC-1065 or a CC-1065analog.

In preferred embodiments of the invention, the cell binding agent is ahumanized anti-CA6 antibody and the cytotoxic agent is a cytotoxic drugsuch as a maytansinoid or a taxane.

More preferably, the cell binding agent is the humanized anti-CA6antibody DS6 and the cytotoxic agent is a maytansine compound, such asDM1 or DM4.

The present invention also includes a method for inhibiting the growthof a cell expressing the CA6 glycotope. In preferred embodiments, themethod for inhibiting growth of the cell expressing the CA6 glycotopetakes place in vivo and results in the death of the cell, although invitro and ex vivo applications are also included.

The present invention also provides a therapeutic composition comprisingthe cytotoxic conjugate, and a pharmaceutically acceptable carrier orexcipient.

The present invention further includes a method of treating a subjecthaving cancer using the therapeutic composition. In preferredembodiments, the cytotoxic conjugate comprises an anti-CA6 antibody anda cytotoxic agent. In more preferred embodiments, the cytotoxicconjugate comprises a humanized DS6 antibody-DM1 conjugate, humanizedDS6 antibody-DM4 or a humanized DS6 antibody-taxane conjugate, and theconjugate is administered along with a pharmaceutically acceptablecarrier or excipient.

The present invention also includes a kit comprising an anti-CA6antibody-cytotoxic agent conjugate and instructions for use. Inpreferred embodiments, the anti-CA6 antibody is the humanized DS6antibody, the cytotoxic agent is a maytansine compound, such as DM1 orDM4, or a taxane, and the instructions are for using the conjugates inthe treatment of a subject having cancer. The kit may also includecomponents necessary for the preparation of a pharmaceuticallyacceptable formulation, such a diluent if the conjugate is in alyophilized state or concentrated form, and for the administration ofthe formulation.

The present invention also includes derivatives of antibodies thatspecifically bind and recognize the CA6 glycotope. In preferredembodiments, the antibody derivatives are prepared by resurfacing orhumanizing antibodies that bind the CA6 glycotope, wherein thederivatives have decreased immunogenicity toward the host.

The present invention further provides for humanized antibodies orfragments thereof that are further labeled for use in research ordiagnostic applications. In preferred embodiments, the label is aradiolabel, a fluorophore, a chromophore, an imaging agent or a metalion.

A method for diagnosis is also provided in which said labeled humanizedantibodies or epitope-binding fragments thereof are administered to asubject suspected of having a cancer, and the distribution of the labelwithin the body of the subject is measured or monitored.

The present invention also provides methods for the treatment of asubject having a cancer by administering a humanized antibody conjugateof the present invention, either alone or in combination with othercytotoxic or therapeutic agents. The cancer can be one or more of, forexample, breast cancer, colon cancer, ovarian carcinoma, endometrialcancer, osteosarcoma, cervical cancer, prostate cancer, lung cancer,synovial carcinoma, pancreatic cancer, a sarcoma or a carcinoma in whichCA6 is expressed or other cancer yet to be determined in which CA6glycotope is expressed predominantly.

Unless otherwise stated, all references and patents cited herein areincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of studies performed to determine the abilityof the DS6 antibody to bind the surface of selected cancer cell lines.The fluorescence of cell lines incubated with the DS6 primary antibodyand FITC conjugated anti-mouse IgG(H+L) secondary antibodies wasmeasured by flow cytometry. The DS6 antibody bound Caov-3 (FIG. 1A) andT-47D (FIG. 1B) cells with an apparent Kd of 1.848 nM and 2.586 nMrespectively. Antigen negative cell lines, SK-OV-3 (FIG. 1C) and Colo205(FIG. 1D) demonstrated no antigen specific binding.

FIG. 2 shows the results of dot blot analysis of epitope expression.Caov-3 (FIG. 2A & FIG. 2B), SKMEL28 (FIG. 2C), and Colo205 (FIG. 2D)cell lysates were individually spotted onto nitrocellulose membranes andthen incubated individually with pronase, proteinase K, neuraminidase orperiodic acid. The membranes were then immunoblotted with the DS6antibody (FIG. 2A), the CM1 antibody (FIG. 2B), the R24 antibody (FIG.2C), or the C242 antibody (FIG. 2D).

FIG. 3 shows the results of a dot blot analysis of DS6 antigenexpression. Caov-3 cell lysates were individually spotted onto PVDFmembranes and then incubated in the presence of trifluoromethanesulfonicacid (TFMSA). The membranes were then immunoblotted with the CM1antibody (1 & 2) or the DS6 antibody (3 & 4).

FIG. 4 shows the results of glycotope analysis of the DS6 antigen.Caov-3 lysates pretreated with N-glycanase (“N-gly”), O-glycanase(“O-gly”), and/or sialidase (“S”) were spotted onto nitrocellulose andthen immunoblotted with the DS6 antibody or the CM1 antibody (Muc-1VNTR).

FIG. 5 shows the results of western blot analysis of the DS6 antigen.Cell lysates were immunoprecipitated (“IP”) and immunoblotted with theDS6 antibody. The antigen corresponds to a >250 kDa protein bandobserved in antigen-positive Caov-3 (FIG. 5A and FIG. 5B) and T47D (FIG.5C) cells. Antigen negative SK-OV-3 (FIG. 5D) and Colo205 (FIG. 5E) celllines do not exhibit this band. After immunoprecipitation, the Protein Gbeads of the Caov-3 cell lysates were incubated with (FIG. 5A)neuraminidase (“N”) or (FIG. 5B) periodic acid (“PA”). Antibody (“α”),pre-IP (“Lys”) and post-IP flow-through (“FT”) lysate controls were runon the same gel. Caov-3 immunoprecipitates were also incubated withN-glycanase (“N-gly”), O-glycanase (“O-gly”), and/or sialidase (“S”)(see FIG. 5F), where the blot was alternatively probed withbiotinylated-DS6 and strepavidin-HRP.

FIG. 6 shows the results of immunoprecipitations and/or immunoblots ofthe DS6 antibody and the CM1 antibody on Caov-3 (FIG. 6A) and HeLa (FIG.6B) cell lysates. Overlapping CM1 and DS6 western blot signals signifythat the DS6 antigen is on the Muc1 protein. In HeLa lysates, the Muc1doublet results from Muc1 expression directed by distinct allelesdiffering in their number of tandem repeats.

FIG. 7 shows a DS6 antibody sandwich ELISA design (FIG. 7A) and astandard curve (FIG. 7B). The standard curve was generated using knownconcentrations of commercially available CA15-3 standards (where 1CA15-3 unit 1 DS6 unit).

FIG. 8 shows quantitative ELISA standard curves. The standard curves ofthe detection antibody (streptavidin-HRP/biotin-DS6) signal (FIG. 8C)were determined using known concentrations of biotin-DS6 either capturedby plated goat anti-mouse IgG (FIG. 8A) or bound directly onto the ELISAplate (FIG. 8B).

FIG. 9 shows the cDNA and amino acid sequences of the light chain (FIG.9A) and heavy chain (FIG. 9B) variable region for the murine DS6antibody. The three CDRs in each sequence are underlined (Kabatdefinitions).

FIG. 10 shows the light (FIG. 10A) and heavy chain (FIG. 10B) CDRs ofthe murine DS6 antibody determined by Kabat definitions. The AbMmodeling software produces a slightly different definition for the heavychain CDRs (FIG. 10 c).

FIG. 11 shows the light chain (“muDS6LC”) (residues 1-95 of SEQ ID NO:7)and heavy chain (“muDS6HC”) (residues 1-98 of SEQ ID NO:9) amino acidsequences for the murine DS6 antibody aligned with the germlinesequences for the IgV_(κ)ap4 (SEQ ID NO:23) and IgVh J558.41 (SEQ IDNO:24) genes. Grey indicates sequence divergence.

FIG. 12 shows the ten light chain and heavy chain antibody sequencesmost homologous to the murine DS6 (muDS6) light chain (“muDS6LC”) andheavy chain (“muDS6HC”) sequences that have solved structure files inthe Brookhaven database. Sequences are aligned in order of most to leasthomologous.

FIG. 13 shows surface accessibility data and calculations to predictwhich framework residues of the murine DS6 antibody light chain variableregion are surface accessible. The positions with 25-35% average surfaceaccessibility are marked (*??*) and were subjected to the second roundanalysis. DS6 antibody light chain variable region (FIG. 13A) and heavychain variable region (FIG. 13B).

FIG. 14 shows the prDS6 v1-0 mammalian expression plasmid map. Thisplasmid was used to build and express the recombinant chimeric andhumanized DS6 antibodies.

FIG. 15 shows amino acid sequences of murine (“muDS6”) and humanized(“huDS6”) (1.01 & 1.21) DS6 antibody light chain (FIG. 15A) and heavychain (FIG. 15B) variable domains.

FIG. 16 shows the cDNA and amino acid sequences of the light chainvariable region for the humanized DS6 antibody (“huDS6”) (1.01 and1.21).

FIG. 17 shows the cDNA and amino acid sequences of the heavy chainvariable region for the humanized DS6 antibody (“huDS6”) 1.01 (FIG. 17A)and 1.21 (FIG. 17B).

FIG. 18 shows flow cytometry binding curves of murine DS6 (muDS6)chimeric DS6 (chDS6), and human DS6 version 1.01 (huDS6 v1.01) andversion huDS6 version 1.21 (huDS6 v1.21) from an assay performed on KBcells. The avidities of the murine, chimeric, and human v1.01 and v1.21DS6 antibodies (muDS6=0.82 nM, chDS6=0.69 nM, huDS6v1.01=0.82 nM andhuDS6v1.21=0.85 nM) are comparable, indicating that resurfacing has notdiminished the avidity.

FIG. 19 shows the results of a competition binding assay of muDS6,chDS6, huDS6 v1.01 and huDS6 v1.21 antibodies with biotinylated muDS6.Varying concentrations of naked muDS6, chDS6, huDS6v1.01 and huDS6v1.21were combined with 2 nM of biotin-muDS6 and the streptavidin-DTAFsecondary. The IC50's (muDS6=1.9 nM, chDS6=1.7 nM, huDS6v1.01=3.0 nM,and huDS6v1.21=1.9 nM) of all antibodies are similar indicating thathumanization has not reduced the avidity.

FIG. 20 shows the results of a determination of the binding affinity ofun-conjugated DS6 antibody versus a DS6 antibody-DM1 conjugate. Theresults demonstrated that DM1 conjugation does not adversely affect thebinding affinity of the antibody. The apparent Kd of the DS6antibody-DM1 conjugate (3.902 nM) (“DS6-DM1”) was slightly greater thanthe naked antibody (2.020 nM) (“DS6”).

FIG. 21 shows the results of an indirect cell viability assay using theDS6 antibody in the presence or absence of the anti-mouse IgG (H+L) DM1conjugate (2° Ab-DM1). Antigen-positive Caov-3 cells were killed in aDS6 antibody-dependent manner (IC₅₀=424.9 pM) only in the presence ofthe secondary conjugate (“DS6+2° Ab-DM1”).

FIG. 22 shows the results of a complement-dependent cytotoxicity (CDC)assay of the muDS6 antibody. The results demonstrate that there is noCDC mediated effect of the DS6 antibody or on HPAC (FIG. 22A) andZR-75-1 (FIG. 22B) cells.

FIG. 23 shows the results of an in vitro cytotoxicity assay of a DS6antibody-DM1 conjugate versus free maytansine. In a clonogenic assay,DS6 antigen-positive ovarian (FIG. 23A), breast (FIG. 23B), cervical(FIG. 23C), and pancreatic (FIG. 23D) cancer cell lines were tested forcytotoxicity of continuous exposure to a DS6 antibody-DM1 conjugate(left panels). These cell lines were similarly tested for maytansinesensitivity by a 72 h exposure to free maytansine (right panels). Theovarian cancer cell lines tested were OVCAR5, TOV-21G, Caov-4 andCaov-3. The breast cancer cell lines tested were T47D, BT-20 and BT-483.The cervical cancer cell lines tested were KB, HeLa and WISH. Thepancreatic cancer cell lines tested were HPAC, Hs766T and HPAF-II.

FIG. 24 shows the results of an in vitro cytotoxicity assay of a DS6antibody-DM1 conjugate. In a MTT cell viability assay, human ovarian(FIG. 24A, FIG. 24B & FIG. 24C), breast (FIG. 24D & FIG. 24E), cervical(FIG. 24F & FIG. 24G), and pancreatic (FIG. 24H & FIG. 241) cancer cellswere killed in a DS6 antibody-DM1 conjugate-dependent manner. Naked DS6did not adversely affect the growth of these cells, indicating that DM1conjugation is required for the cytotoxicity.

FIG. 25A shows the results of an in vivo anti-tumor efficacy study of aDS6 antibody-DM1 conjugate on established subcutaneous KB tumorxenografts. Tumor cells were inoculated on day 0, and the firsttreatment was given on day 6. Immunoconjugate treatments continued dailyfor a total of 5 doses. PBS control animals were euthanized once tumorvolumes exceeded 1500 mm³. The conjugate was given at a dose of 150 or225 μg/kg DM1, corresponding to antibody concentrations of 5.7 and 8.5mg/kg respectively. The body weights (FIG. 25B) of the mice weremonitored during the course of the study.

FIG. 26 shows the results of an antitumor efficacy study of a DS6antibody-DM1 conjugate on established subcutaneous tumor xenografts.OVCAR5 (FIG. 26A and FIG. 26B), TOV-21G (FIG. 26C and FIG. 26D), HPAC(FIG. 26E and FIG. 26F), and HeLa (FIG. 26G and FIG. 26H) cells wereinoculated on day 0, and immunoconjugate treatments were given on day 6and 13. PBS control animals were euthanized once tumor volumes exceeded1000 mm³. The conjugate was given at a dose of 600 μg/kg DM1,corresponding to an antibody concentration 27.7 mg/kg. Tumor volume(FIG. 26A, FIG. 26C, FIG. 26E, and FIG. 26G) and body weight (FIG. 26B,FIG. 26D, FIG. 26F, and FIG. 26H) of the mice were monitored during thecourse of the study.

FIG. 27 shows the results of an in vivo efficacy study of a muDS6antibody-DM1 conjugate on intraperitoneal OVCAR5 tumors. Tumor cellswere injected intraperitoneally on day 0, and immunoconjugate treatmentswere given on day 6 and 13. Animals were euthanized once body weightloss exceeded 20%.

FIG. 28 shows the flow cytometry binding curve from a study of thebinding affinity of naked and taxane-conjugated DS6 antibody on HeLacells. Taxane (MM1-202)-conjugation does not adversely affect thebinding affinity of the antibody. The apparent Kd of the DS6-MM1-202conjugate (1.24 nM) was slightly greater than the naked DS6 antibody(620 pM).

FIG. 29 shows in vitro binding and potency of humanized DS6 version 1.01antibody conjugate. Conjugation of huDS6v1.01 with DM4 has little effecton the avidity of huDS6v1.01 for KB cells (FIG. 29A). huDS6v1.01-DM4shows potent in vitro cytotoxicity toward DS6-expressing WISH cells withan IC₅₀ of 0.44 nM (FIG. 29B).

FIG. 30 shows the results of an in vivo efficacy study withhuDS6v1.01-DM4 conjugate in an HPAC pancreatic cancer model.huDS6v1.01-DM4 showed potent anti-tumor activity whereas the B4-DM4control conjugate whose target is not expressed in the HPAC model hadessentially no activity (FIG. 30A). The administered dose of 200 μg/kgwas not toxic to the animals as indicated by the lack of weight loss(FIG. 30B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other features, anti-CA6monoclonal antibodies, anti-CA6 humanized antibodies, and fragments ofthe anti-CA6 antibodies. Each of the antibodies and antibody fragmentsof the present invention are designed to specifically recognize and bindthe CA6 glycotope on the surface of a cell. CA6 is known to be expressedby many human tumors: 95% of serous ovarian carcinomas, 50% ofendometrioid ovarian carcinomas, 50% of the neoplasms of the uterinecervix, 69% of the neoplasms of the endometrius, 80% of neoplasms of thevulva, 60% of breast carcinomas, 67% pancreatic tumors, and 48% oftumors of the urothelium, but is rarely expressed by normal humantissue.

A report by Kearse et al., Int. J. Cancer 88(6):866-872 (2000)misidentified the protein on which the CA6 epitope is found as an 80 kDaprotein having an N-linked carbohydrate containing the CA6 epitope whenthey used a hybridoma supernatant to characterize it. Using purified DS6we have since demonstrated that the CA6 epitope is found on an O-linkedcarbohydrate of a greater than 250 kDa non-disulfide-linkedglycoprotein. Furthermore, the glycoprotein was identified as the mucin,Muc1. Because different Muc1 alleles have varying numbers of tandemrepeats in the variable number tandem repeat (VNTR) domain cells oftenexpress two distinct Muc1 proteins of different size(Taylor-Papadimitriou, Biochim. Biophys. Acta 1455(2-3):301-13 (1999).Because of differences in the number of repeats in the VNTR domain aswell as differences in glycosylation the molecular weight of Muc1 variesfrom cell line to cell line.

The susceptibility of CA6 immunoreactivity to periodic acid indicatesCA6 is a carbohydrate epitope “glycotope.” The additional susceptibilityof CA6 immunoreactivity to treatment with neuraminidase from Vibriocholerae indicates that the CA6 epitope is a sialic acid dependentglycotope, thus a “sialoglycotope.”

Details of the characterization of CA6 can be found in the Example 2(see below). Additional details on CA6 may be found in WO 02/16401;Wennerberg et al., Am. J. Pathol. 143(4):1050-1054 (1993); Smith et al.,Human Antibodies 9:61-65 (1999); Kearse et al., Int. J. Cancer88(6):866-872 (2000); Smith et al., Int. J. Gynecol. Pathol. 20(3):260-6(2001); and Smith et al., Appl. Immunohistochem. Mol. Morphol.10(2):152-8 (2002).

The present invention also includes cytotoxic conjugates comprising twoprimary components. The first component is a cell binding agent thatrecognizes and binds the CA6 glycotope. The cell binding agent shouldrecognize the CA6 sialoglycotope on Muc 1 with a high degree ofspecificity so that the cytotoxic conjugates recognize and bind only thecells for which they are intended. A high degree of specificity willallow the conjugates to act in a targeted fashion with littleside-effects resulting from non-specific binding.

In another embodiment, the cell binding agent of the present inventionalso recognizes the CA6 glycotope with a high degree of affinity so thatthe conjugates will be in contact with the target cell for a sufficientperiod of time to allow the cytotoxic drug portion of the conjugate toact on the cell, and/or to allow the conjugates sufficient time in whichto be internalized by the cell.

In a preferred embodiment, the cytotoxic conjugates comprise an anti-CA6antibody as the cell binding agent, more preferably the murine DS6anti-CA6 monoclonal antibody. In a more preferred embodiment, thecytotoxic conjugates comprises a humanized DS6 antibody or anepitope-binding fragment thereof. The DS6 antibody is able to recognizeCA6 with a high degree of specificity and directs the cytotoxic agent toan abnormal cell or a tissue, such as cancer cells, in a targetedfashion.

The second component of the cytotoxic conjugates of the presentinvention is a cytotoxic agent. In preferred embodiments, the cytotoxicagent is a taxol, a maytansinoid such as DM1 or DM4, CC-1065 or aCC-1065 analog. In preferred embodiments, the cell binding agents of thepresent invention are covalently attached, directly or via a cleavableor non-cleavable linker, to the cytotoxic agent.

The cell binding agents, cytotoxic agents, and linkers are discussed inmore detail below.

Cell Binding Agents

The effectiveness of the compounds of the present invention astherapeutic agents depends on the careful selection of an appropriatecell binding agent. Cell binding agents may be of any kind presentlyknown, or that become known and includes peptides and non-peptides. Thecell binding agent may be any compound that can bind a cell, either in aspecific or non-specific manner. Generally, these can be antibodies(especially monoclonal antibodies), lymphokines, hormones, growthfactors, vitamins, nutrient-transport molecules (such as transferrin),or any other cell binding molecule or substance.

More specific examples of cell binding agents that can be used include:

(a) polyclonal antibodies;

(b) monoclonal antibodies;

(c) fragments of antibodies such as Fab, Fab′, and F(ab′)₂, Fv (Parham,J. Immunol. 131:2895-2902 (1983); Spring et al. J. Immunol. 113:470-478(1974); Nisonoff et al. Arch. Biochem. Biophys. 89:230-244 (1960));

(d) interferons (e.g. alpha., .beta., gamma.);

(e) lymphokines such as IL-2, IL-3, IL-4, IL-6;

(f) hormones such as insulin, TRH (thyrotropin releasing hormone), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens;

(g) growth factors and colony-stimulating factors such as EGF,TGF-alpha, FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today5:155-158 (1984));

(h) transferrin (O'Keefe et al. J. Biol. Chem. 260:932-937 (1985)); and

(i) vitamins, such as folate.

Antibodies

Selection of the appropriate cell binding agent is a matter of choicethat depends upon the particular cell population that is to be targeted,but in general, antibodies are preferred if an appropriate one isavailable or can be prepared, more preferably a monoclonal antibody.

Monoclonal antibody techniques allow for the production of extremelyspecific cell binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.Sensitized human cells can also be used. Another method of creatingmonoclonal antibodies is the use of phage libraries of scFv (singlechain variable region), specifically human scFv (see e.g., Griffiths etal., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO92/01047; Liming et al., WO 99/06587).

A typical antibody is comprised of two identical heavy chains and twoidentical light chains that are joined by disulfide bonds. The variableregion is a portion of the antibody heavy chains and light chains thatdiffers in sequence among antibodies and that cooperates in the bindingand specificity of each particular antibody for its antigen. Variabilityis not usually evenly distributed throughout antibody variable regions.It is typically concentrated within three segments of a variable regioncalled complementarity-determining regions (CDRs) or hypervariableregions, both in the light chain and the heavy chain variable regions.The more highly conserved portions of the variable regions are calledthe framework regions. The variable regions of heavy and light chainscomprise four framework regions, largely adopting a beta-sheetconfiguration, with each framework region connected by the three CDRs,which form loops connecting the beta-sheet structure, and in some casesforming part of the beta-sheet structure. The CDRs in each chain areheld in close proximity by the framework regions and, with the CDRs fromthe other chain, contribute to the formation of the antigen binding siteof antibodies (E. A. Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition, 1991, NIH).

The constant region is a portion of the heavy chain. While not involveddirectly in binding an antibody to an antigen, it does exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

A suitable monoclonal antibody for use in the present invention includesthe murine DS6 monoclonal antibody (U.S. Pat. No. 6,596,503; ATCCdeposit number PTA-4449).

Humanized or Resurfaced DS6 Antibodies

Preferably, a humanized anti-CA6 antibody is used as the cell bindingagent of the present invention. A preferred embodiment of such ahumanized antibody is a humanized DS6 antibody, or an epitope-bindingfragment thereof.

The goal of humanization is a reduction in the immunogenicity of axenogenic antibody, such as a murine antibody, for introduction into ahuman, while maintaining the full antigen binding affinity andspecificity of the antibody.

Humanized antibodies may be produced using several technologies such asresurfacing and CDR grafting. As used herein, the resurfacing technologyuses a combination of molecular modeling, statistical analysis andmutagenesis to alter the non-CDR surfaces of antibody variable regionsto resemble the surfaces of known antibodies of the target host.

Strategies and methods for the resurfacing of antibodies, and othermethods for reducing immunogenicity of antibodies within a differenthost, are disclosed in U.S. Pat. No. 5,639,641 (Pedersen et al.), whichis hereby incorporated in its entirety by reference. Briefly, in apreferred method, (1) position alignments of a pool of antibody heavyand light chain variable regions is generated to give a set of heavy andlight chain variable region framework surface exposed positions whereinthe alignment positions for all variable regions are at least about 98%identical; (2) a set of heavy and light chain variable region frameworksurface exposed amino acid residues is defined for a rodent antibody (orfragment thereof); (3) a set of heavy and light chain variable regionframework surface exposed amino acid residues that is most closelyidentical to the set of rodent surface exposed amino acid residues isidentified; (4) the set of heavy and light chain variable regionframework surface exposed amino acid residues defined in step (2) issubstituted with the set of heavy and light chain variable regionframework surface exposed amino acid residues identified in step (3),except for those amino acid residues that are within 5 Å of any atom ofany residue of the complementarity-determining regions of the rodentantibody; and (5) the humanized rodent antibody having bindingspecificity is produced.

Antibodies can be humanized using a variety of other techniquesincluding CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos.5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0519 596; Padlan E. A., 1991, Molecular Immunology 28(4/5):489-498;Studnicka G. M. et al., 1994, Protein Engineering 7(6):805-814; RoguskaM. A. et al., 1994, PNAS 91:969-973), and chain shuffling (U.S. Pat. No.5,565,332). Human antibodies can be made by a variety of methods knownin the art including phage display methods. See also U.S. Pat. Nos.4,444,887, 4,716,111, 5,545,806, and 5,814,318; and international patentapplication publication numbers WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 (said referencesincorporated by reference in their entireties).

In preferred embodiment, the present invention provides humanizedantibodies or fragments thereof that recognizes a novel sialoglycotope(the CA6 glycotope) on the Muc1 mucin. In another embodiment, thehumanized antibodies or epitope-binding fragments thereof have theadditional ability to inhibit growth of a cell expressing the CA6glycotope.

In more preferred embodiments, there are provided resurfaced orhumanized versions of the DS6 antibody wherein surface-exposed residuesof the antibody or its fragments are replaced in both light and heavychains to more closely resemble known human antibody surfaces. Thehumanized DS6 antibodies or epitope-binding fragments thereof of thepresent invention have improved properties. For example, humanized DS6antibodies or epitope-binding fragments thereof specifically recognize anovel sialoglycotope (the CA6 glycotope) on the Muc1 mucin. Morepreferably, the humanized DS6 antibodies or epitope-binding fragmentsthereof have the additional ability to inhibit growth of a cellexpressing the CA6 glycotope. The humanized antibody or anepitope-binding fragment thereof can be conjugated to a drug, such as amaytansinoid, to form a prodrug having specific cytotoxicity towardsantigen-expressing cells by targeting the drug to the novel Muc1sialoglycotope, CA6. Cytotoxic conjugates comprising such antibodies anda small, highly toxic drug (e.g., maytansinoids, taxanes, and CC-1065analogs) can be used as a therapeutic for treatment of tumors, such asbreast and ovarian tumors.

The humanized versions of the DS6 antibody are also fully characterizedherein with respect to their respective amino acid sequences of bothlight and heavy chain variable regions, the DNA sequences of the genesfor the light and heavy chain variable regions, the identification ofthe CDRs, the identification of their surface amino acids, anddisclosure of a means for their expression in recombinant form.

In one embodiment, there is provided a humanized antibody orepitope-binding fragment thereof having a heavy chain including CDRshaving amino acid sequences represented by SEQ ID NOs:1-3: (SEQ IDNO: 1) S Y N M H (SEQ ID NO: 2) Y I Y P G N G A T N Y N Q K F K G (SEQID NO: 3) G D S V P F A Y

When the heavy chain CDRs are determined by the AbM modeling softwarethey are represented by SEQ ID NOs:20-22: (SEQ ID NO: 20) G Y T F T S YN M H (SEQ ID NO: 21) Y I Y P G N G A T N (SEQ ID NO: 22) G D S V P F AY

In the same embodiment, the humanized antibody or epitope-bindingfragment thereof has a light chain that comprises CDRs having amino acidsequences represented by SEQ ID NOS:4-6: (SEQ ID NO: 4) S A H S S V S FM H (SEQ ID NO: 5) S T S S L A S (SEQ ID NO: 6) Q Q R S S F P L T

Also provided are humanized antibodies and epitope-binding fragmentsthereof having a light chain variable region that has an amino acidsequence that shares at least 90% sequence identity with an amino acidsequence represented by SEQ ID NO:7 or SEQ ID NO:8: (SEQ ID NO: 7)QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAG TKLELKR. (SEQ ID NO:8) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAG TKLELKR.

Similarly, there are provided humanized antibodies and epitope-bindingfragments thereof having a heavy chain variable region that has an aminoacid sequence that shares at least 90% sequence identity with an aminoacid sequence represented by SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11:(SEQ ID NO: 9) QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.(SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.(SEQ ID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.

In another embodiment, humanized antibodies and epitope-bindingfragments thereof are provided having a humanized or resurfaced lightchain variable region having an amino acid sequence corresponding to SEQID NO: 8 (SEQ ID NO: 8)EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYSTSSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAG TKLELKR.

Similarly, humanized antibodies and epitope-binding fragments thereofare provided having a humanized or resurfaced heavy chain variableregion having an amino acid sequence corresponding to SEQ ID NO: 10 orSEQ ID NO:11: (SEQ ID NO: 10)QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.(SEQ ID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGYIYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.

The humanized antibodies and epitope-binding fragments thereof of thepresent invention can also include versions of light and/or heavy chainvariable regions in which human surface amino acid residues in proximityto the CDRs are replaced by the corresponding muDS6 surface residues atone or more positions defined by the residues in Table 1 (Kabatnumbering) marked with an asterisk in order to retain the bindingaffinity and specificity of muDS6. TABLE 1 muDS6 framework residuesproximal to a CDR (Kabat numbering) Light chain Heavy chain Q1* Q1 V3K64* T5 P73* P40 S74 G57 A60 S67 E81

The primary amino acid and DNA sequences of the DS6 antibody light andheavy chains, and of humanized versions thereof, are disclosed herein.However, the scope of the present invention is not limited to antibodiesand fragments comprising these sequences. Instead, all antibodies andfragments that specifically bind to CA6 as a unique tumor-specificglycotope on the Muc 1 receptor are included in the present invention.Preferably, the antibodies and fragments that specifically bind to CA6also antagonize the biological activity of the receptor. Morepreferably, such antibodies further are substantially devoid of agonistactivity. Thus, antibodies and antibody fragments of the presentinvention may differ from the DS6 antibody or the humanized derivativesthereof, in the amino acid sequences of their scaffold, CDRs, and/orlight chain and heavy chain, and still fall within the scope of thepresent invention.

The CDRs of the DS6 antibody are identified by modeling and theirmolecular structures have been predicted. Again, while the CDRs areimportant for epitope recognition, they are not essential to theantibodies and fragments of the invention. Accordingly, antibodies andfragments are provided that have improved properties produced by, forexample, affinity maturation of an antibody of the present invention.

The mouse light chain IgV_(κ) ap4 germline gene and heavy chain IgVhJ558.41 germline gene from which DS6 was likely derived are shown inFIG. 11 aligned with the sequence of the DS6 antibody. The comparisonidentifies probable somatic mutations in the DS6 antibody, includingseveral in the CDRs.

The sequence of the heavy chain and light chain variable region of theDS6 antibody, and the sequences of the CDRs of the DS6 antibody were notpreviously known and are set forth in FIGS. 9A and 9B. Such informationcan be used to produce humanized versions of the DS6 antibody.

Antibody Fragments

The antibodies of the present invention include both the full lengthantibodies discussed above, as well as epitope-binding fragments. Asused herein, “antibody fragments” include any portion of an antibodythat retains the ability to bind to the epitope recognized by the fulllength antibody, generally termed “epitope-binding fragments.” Examplesof antibody fragments include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (dsFv) and fragments comprising either a V_(L) orV_(H) region. Epitope-binding fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, C_(H)1,C_(H)2, and C_(H)3 domains.

Such fragments may contain one or both Fab fragments or the F(ab′)₂fragment. Preferably, the antibody fragments contain all six CDRs of thewhole antibody, although fragments containing fewer than all of suchregions, such as three, four or five CDRs, are also functional. Further,the fragments may be or may combine members of any one of the followingimmunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclassesthereof.

Fab and F(ab′)₂ fragments may be produced by proteolytic cleavage, usingenzymes such as papain (Fab fragments) or pepsin (F(ab′)₂ fragments).

The single-chain FVs (scFvs) fragments are epitope-binding fragmentsthat contain at least one fragment of an antibody heavy chain variableregion (V_(H)) linked to at least one fragment of an antibody lightchain variable region (V_(L)). The linker may be a short, flexiblepeptide selected to assure that the proper three-dimensional folding ofthe (V_(L)) and (V_(H)) regions occurs once they are linked so as tomaintain the target molecule binding-specificity of the whole antibodyfrom which the single-chain antibody fragment is derived. The carboxylterminus of the (V_(L)) or (V_(H)) sequence may be covalently linked bya linker to the amino acid terminus of a complementary (V_(L)) or(V_(H)) sequence.

Single-chain antibody fragments of the present invention contain aminoacid sequences having at least one of the variable or complementaritydetermining regions (CDRs) of the whole antibodies described in thisspecification, but are lacking some or all of the constant domains ofthose antibodies. These constant domains are not necessary for antigenbinding, but constitute a major portion of the structure of wholeantibodies. Single-chain antibody fragments may therefore overcome someof the problems associated with the use of antibodies containing a partor all of a constant domain. For example, single-chain antibodyfragments tend to be free of undesired interactions between biologicalmolecules and the heavy-chain constant region, or other unwantedbiological activity. Additionally, single-chain antibody fragments areconsiderably smaller than whole antibodies and may therefore havegreater capillary permeability than whole antibodies, allowingsingle-chain antibody fragments to localize and bind to targetantigen-binding sites more efficiently. Also, antibody fragments can beproduced on a relatively large scale in prokaryotic cells, thusfacilitating their production. Furthermore, the relatively small size ofsingle-chain antibody fragments makes them less likely to provoke animmune response in a recipient than whole antibodies.

Single-chain antibody fragments may be generated by molecular cloning,antibody phage display library or similar techniques well known to theskilled artisan. These proteins may be produced, for example, ineukaryotic cells or prokaryotic cells, including bacteria. Theepitope-binding fragments of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, such phage can be utilized to display epitope-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an epitope-binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage including fd and M13 binding domains expressed from phage withFab, Fv or disulfide-stabilized Fv antibody domains recombinantly fusedto either the phage gene III or gene VIII protein.

Examples of phage display methods that can be used to make theepitope-binding fragments of the present invention include thosedisclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ameset al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al.,1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18;Burton et al., 1994, Advances in Immunology 57:191-280; PCT applicationNo. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;5,658,727; 5,733,743 and 5,969,108; each of which is incorporated hereinby reference in its entirety.

After phage selection, the regions of the phage encoding the fragmentscan be isolated and used to generate the epitope-binding fragmentsthrough expression in a chosen host, including mammalian cells, insectcells, plant cells, yeast, and bacteria, using recombinant DNAtechnology, e.g., as described in detail below. For example, techniquesto recombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., 1992, BioTechniques12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043; said references incorporated by referencein their entireties. Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology203:46-88; Shu et al., 1993, PNAS 90:7995-7999; Skerra et al., 1988,Science 240:1038-1040.

Functional Equivalents

Also included within the scope of the invention are functionalequivalents of the anti-CA6 antibody and the humanized anti-CA6antibody. The term “functional equivalents” includes antibodies withhomologous sequences, chimeric antibodies, artificial antibodies andmodified antibodies, for example, wherein each functional equivalent isdefined by its ability to bind to CA6. The skilled artisan willunderstand that there is an overlap in the group of molecules termed“antibody fragments” and the group termed “functional equivalents.”Methods of producing functional equivalents are disclosed, for example,in PCT Application WO 93/21319, European Patent Application No. 239,400;PCT Application WO 89/09622; European Patent Application 338,745; andEuropean Patent Application EP 332,424, which are incorporated in theirrespective entireties by reference.

Antibodies with homologous sequences are those antibodies with aminoacid sequences that have sequence homology with amino acid sequence ofan anti-CA6 antibody and a humanized anti-CA6 antibody of the presentinvention. Preferably homology is with the amino acid sequence of thevariable regions of the anti-CA6 antibody and humanized anti-CA6antibody of the present invention. “Sequence homology” as applied to anamino acid sequence herein is defined as a sequence with at least about90%, 91%, 92%, 93%, or 94% sequence homology, and more preferably atleast about 95%, 96%, 97%, 98%, or 99% sequence homology to anotheramino acid sequence, as determined, for example, by the FASTA searchmethod in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA85, 2444-2448 (1988).

As used herein, a chimeric antibody is one in which different portionsof an antibody are derived from different animal species. For example,an antibody having a variable region derived from a murine monoclonalantibody paired with a human immunoglobulin constant region. Methods forproducing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies etal., 1989, J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entireties.

Humanized forms of chimeric antibodies are made by substituting thecomplementarity determining regions of, for example, a mouse antibody,into a human framework domain, e.g., see PCT Pub. No. W092/22653.Humanized chimeric antibodies preferably have constant regions andvariable regions other than the complementarity determining regions(CDRs) derived substantially or exclusively from the corresponding humanantibody regions and CDRs derived substantially or exclusively from amammal other than a human.

Artificial antibodies include scFv fragments, diabodies, triabodies,tetrabodies and mru (see reviews by Winter, G. and Milstein, C., 1991,Nature 349: 293-299; Hudson, P. J., 1999, Current Opinion in Immunology11: 548-557), each of which has antigen-binding ability. In the singlechain Fv fragment (scFv), the V_(H) and V_(L) domains of an antibody arelinked by a flexible peptide. Typically, this linker peptide is about 15amino acid residues long. If the linker is much smaller, for example 5amino acids, diabodies are formed, which are bivalent scFv dimers. Ifthe linker is reduced to less than three amino acid residues, trimericand tetrameric structures are formed that are called triabodies andtetrabodies. The smallest binding unit of an antibody is a CDR,typically the CDR2 of the heavy chain which has sufficient specificrecognition and binding that it can be used separately. Such a fragmentis called a molecular recognition unit or mru. Several such mrus can belinked together with short linker peptides, therefore forming anartificial binding protein with higher avidity than a single mru.

The functional equivalents of the present application also includemodified antibodies, e.g., antibodies modified by the covalentattachment of any type of molecule to the antibody. For example,modified antibodies include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Thecovalent attachment does not prevent the antibody from generating ananti-idiotypic response. These modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the modified antibodies may contain one or morenon-classical amino acids.

Functional equivalents may be produced by interchanging different CDRson different chains within different frameworks. Thus, for example,different classes of antibody are possible for a given set of CDRs bysubstitution of different heavy chains, whereby, for example, IgG1-4,IgM, IgA1-2, IgD, IgE antibody types and isotypes may be produced.Similarly, artificial antibodies within the scope of the invention maybe produced by embedding a given set of CDRs within an entirelysynthetic framework.

Functional equivalents may be readily produced by mutation, deletionand/or insertion within the variable and/or constant region sequencesthat flank a particular set of CDRs, using a wide variety of methodsknown in the art.

The antibody fragments and functional equivalents of the presentinvention encompass those molecules with a detectable degree of bindingto CA6, when compared to the DS6 antibody. A detectable degree ofbinding includes all values in the range of at least 10-100%, preferablyat least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%,95% or 99% the binding ability of the murine DS6 antibody to CA6.

Improved Antibodies

The CDRs are of primary importance for epitope recognition and antibodybinding. However, changes may be made to the residues that comprise theCDRs without interfering with the ability of the antibody to recognizeand bind its cognate epitope. For example, changes that do not affectepitope recognition, yet increase the binding affinity of the antibodyfor the epitope may be made.

Thus, also included in the scope of the present invention are improvedversions of both the murine and humanized antibodies, which alsospecifically recognize and bind CA6, preferably with increased affinity.

Several studies have surveyed the effects of introducing one or moreamino acid changes at various positions in the sequence of an antibody,based on the knowledge of the primary antibody sequence, on itsproperties such as binding and level of expression (Yang, W. P. et al.,1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc. Natl.Acad. Sci. USA, 95, 8910-8915; Vaughan, T. J. et al., 1998, NatureBiotechnology, 16, 535-539).

In these studies, equivalents of the primary antibody have beengenerated by changing the sequences of the heavy and light chain genesin the CDR1, CDR2, CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E.coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16, 535-539;Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “Phage Display ofPeptides and Proteins”, Eds. Kay, B. K. et al., Academic Press). Thesemethods of changing the sequence of the primary antibody have resultedin improved affinities of the secondary antibodies (Gram, H. et al.,1992, Proc. Natl. Acad. Sci. USA, 89, 3576-3580; Boder, E. T. et al.,2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705; Davies, J. andRiechmann, L., 1996, Immunotechnolgy, 2, 169-179; Thompson, J. et al.,1996, J. Mol. Biol., 256, 77-88; Short, M. K. et al., 2002, J. Biol.Chem., 277, 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276,27622-27628).

By a similar directed strategy of changing one or more amino acidresidues of the antibody, the antibody sequences described in thisinvention can be used to develop anti-CA6 antibodies with improvedfunctions, including improved affinity for CA6.

Improved antibodies also include those antibodies having improvedcharacteristics that are prepared by the standard techniques of animalimmunization, hybridoma formation and selection for antibodies withspecific characteristics

Cytotoxic Agents

The cytotoxic agent used in the cytotoxic conjugate of the presentinvention may be any compound that results in the death of a cell, orinduces cell death, or in some manner decreases cell viability.Preferred cytotoxic agents include, for example, maytansinoids andmaytansinoid analogs, taxoids, CC-1065 and CC-1065 analogs, dolastatinand dolastatin analogs, defined below. These cytotoxic agents areconjugated to the antibodies, antibodies fragments, functionalequivalents, improved antibodies and their analogs as disclosed herein

The cytotoxic conjugates may be prepared by in vitro methods. In orderto link a drug or prodrug to the antibody, a linking group is used.Suitable linking groups are well known in the art and include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred linkinggroups are disulfide groups and thioether groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between the antibody and the drug or prodrug.

Maytansinoids

Among the cytotoxic agents that may be used in the present invention toform a cytotoxic conjugate, are maytansinoids and maytansinoid analogs.Examples of suitable maytansinoids include maytansinol and maytansinolanalogs. Maytansinoids are drugs that inhibit microtubule formation andthat are highly toxic to mammalian cells.

Examples of suitable maytansinol analogues include those having amodified aromatic ring and those having modifications at otherpositions. Such suitable maytansinoids are disclosed in U.S. Pat. Nos.4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Specific examples of suitable analogues of maytansinol having a modifiedaromatic ring include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamytocin P2);

(2) C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable analogues of maytansinol havingmodifications of other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H₂S or P₂S₅);

(2) C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudiflora);

(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

In a preferred embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoid (DM1), formallytermed N^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine, asthe cytotoxic agent. DM1 is represented by the following structuralformula (I):

In another preferred embodiment, the cytotoxic conjugates of the presentinvention utilize the thiol-containing maytansinoidN^(2′)-deacetyl-N-^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine asthe cytotoxic agent. DM4 is represented by the following structuralformula (II):

In further embodiments of the invention, other maytansines, includingthiol and disulfide-containing maytansinoids bearing a mono or di-alkylsubstitution on the carbon atom bearing the sulfur atom, may be used.These include a maytansinoid having, at C-3, C-14 hydroxymethyl, C-15hydroxy, or C-20 desmethyl, an acylated amino acid side chain with anacyl group bearing a hindered sulfhydryl group, wherein the carbon atomof the acyl group bearing the thiol functionality has one or twosubstituents, said substituents being CH3, C2H5, linear or branchedalkyl or alkenyl having from 1 to 10 carbon atoms, cyclic alkyl oralkenyl having from 3 to 10 carbon atoms, phenyl, substituted phenyl, orheterocyclic aromatic or heterocycloalkyl radical, and further whereinone of the substituents can be H, and wherein the acyl group has alinear chain length of at least three carbon atoms between the carbonylfunctionality and the sulfur atom.

Such additional maytansines include compounds represented by formula(III):

wherein:Y′ represents(CR₇CR₈)_(l)(CR₉═CR₁₀)_(p)C═C_(q)A_(r)(CR₅CR₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C═C)_(s)B_(t)(CR₃CR₄)_(n)CR₁R₂SZ,wherein:

R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenylhaving from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenylhaving from 3 to 10 carbon atoms, phenyl, substituted phenyl orheterocyclic aromatic or heterocycloalkyl radical, and in addition R₂can be H;

A, B, D are cycloalkyl or cycloalkenyl having 3-10 carbon atoms, simpleor substituted aryl or heterocyclic aromatic or heterocycloalkylradical;

R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, and R₁₂ are each independently H, CH₃,C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branchedor cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

l, m, n, o, p, q, r, s, and t are each independently 0 or an integer offrom 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and tare not zero at any one time; and

Z is H, SR or —COR, wherein R is linear alkyl or alkenyl having from 1to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to10 carbon atoms, or simple or substituted aryl or heterocyclic aromaticor heterocycloalkyl radical.

Preferred embodiments of formula (III) include compounds of formula(III) wherein:

R₁ is H, R₂ is methyl and Z is H.

R₁ and R₂ are methyl and Z is H.

R₁ is H, R₂ is methyl, and Z is —SCH₃.

R₁ and R₂ are methyl, and Z is —SCH₃.

Such additional maytansines also include compounds represented byformula (IV-L), (IV-D), or (IV-D,L):

wherein:

Y represents (CR₇CR₈)_(l)(CR₅CR₆)_(m)(CR₃CR₄)_(n)CR₁R₂SZ,

wherein:

R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenylhaving from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenylhaving from 3 to 10 carbon atoms, phenyl, substituted phenyl, orheterocyclic aromatic or heterocycloalkyl radical, and in addition R₂can be H;

R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅, linearalkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclicalkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substitutedphenyl, or heterocyclic aromatic or heterocycloalkyl radical;

l, m and n are each independently an integer of from 1 to 5, and inaddition n can be 0;

-   Z is H, SR or —COR wherein R is linear or branched alkyl or alkenyl    having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl having    from 3 to 10 carbon atoms, or simple or substituted aryl or    heterocyclic aromatic or heterocycloalkyl radical; and

May represents a maytansinoid which bears the side chain at C-3, C-14hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Preferred embodiments of formulas (IV-L), (IV-D) and (IV-D,L) includecompounds of formulas (IV-L), (IV-D) and (IV-D,L) wherein:

R₁ is H, R₂ is methyl, R₅, R₆, R₇, and R₈ are each H, l and m are each1, n is 0, and Z is H.

R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1, n is 0,and Z is H.

R₁ is H, R₂ is methyl, R₅, R₆, R₇, and R₈ are each H, l and m are each1, n is 0, and Z is —SCH₃.

R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1, n is 0,and Z is —SCH₃.

Preferably the cytotoxic agent is represented by formula (IV-L).

Such additional maytansines also include compounds represented byformula (V):

wherein:

Y represents (CR₇CR₈)_(l)(CR₅CR₆)_(m)(CR₃CR₄)_(n)CR₁R₂SZ,

wherein:

R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenylhaving from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenylhaving from 3 to 10 carbon atoms, phenyl, substituted phenyl orheterocyclic aromatic or heterocycloalkyl radical, and in addition R₂can be H;

R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅, linearalkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclicalkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substitutedphenyl, or heterocyclic aromatic or heterocycloalkyl radical;

l, m and n are each independently an integer of from 1 to 5, and inaddition n can be 0; and

Z is H, SR or —COR, wherein R is linear alkyl or alkenyl having from 1to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3 to10 carbon atoms, or simple or substituted aryl or heterocyclic aromaticor heterocycloalkyl radical.

Preferred embodiments of formula (V) include compounds of formula (V)wherein:

R1 is H, R2 is methyl, R5, R6, R7, and R8 are each H; l and m are each1; n is 0; and Z is H.

R₁ and R₂ are methyl; R₅, R₆, R₇, R₈ are each H, l and m are 1; n is 0;and Z is H.

R₁ is H, R₂ is methyl, R₅, R₆, R₇, and R₈ are each H, l and m are each1, n is 0, and Z is —SCH₃.

R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1, n is 0,and Z is —SCH₃.

Such additional maytansines further include compounds represented byformula (VI-L), (VI-D), or (VI-D,L):

wherein:Y₂ represents (CR₇CR₈)_(l)(CR₅CR₆)_(m)(CR₃CR₄)_(n)CR₁R₂SZ₂,wherein:

R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenylhaving from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenylhaving from 3 to 10 carbon atoms, phenyl, substituted phenyl orheterocyclic aromatic or heterocycloalkyl radical, and in addition R₂can be H;

R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅, linearcyclic alkyl or alkenyl having from 1 to 10 carbon atoms, branched orcyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

l, m and n are each independently an integer of from 1 to 5, and inaddition n can be 0;

-   Z₂ is SR or COR, wherein R is linear alkyl or alkenyl having from 1    to 10 carbon atoms, branched or cyclic alkyl or alkenyl having from    3 to 10 carbon atoms, or simple or substituted aryl or heterocyclic    aromatic or heterocycloalkyl radical; and

May is a maytansinoid.

Such additional maytansines also include compounds represented byformula (VII):

wherein:Y₂′ represents(CR₇CR₈)_(l)(CR₉═CR₁₀)_(p)(C═C)_(q)A_(r)(CR₅CR₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C═C)_(s)B_(t)(CR₃CR₄)_(n)CR₁R₂SZ₂,wherein:

R₁ and R₂ are each independently CH₃, C₂H₅, linear branched or alkyl oralkenyl having from 1 to 10 carbon atoms, cyclic alkyl or alkenyl havingfrom 3 to 10 carbon atoms, phenyl, substituted phenyl or heterocyclicaromatic or heterocycloalkyl radical, and in addition R₂ can be H;

A, B, and D each independently is cycloalkyl or cycloalkenyl having 3 to10 carbon atoms, simple or substituted aryl, or heterocyclic aromatic orheterocycloalkyl radical;

R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, and R₁₂ are each independently H, CH₃,C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branchedor cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;

l, m, n, o, p, q, r, s, and t are each independently 0 or an integer offrom 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and tare not zero at any one time; and

Z₂ is SR or —COR, wherein R is linear alkyl or alkenyl having from 1 to10 carbon atoms, branched or cyclic alkyl or alkenyl having from 3-10carbon atoms, or simple or substituted aryl or heterocyclic aromatic orheterocycloalkyl radical.

Preferred embodiments of formula (VII) include compounds of formula(VII) wherein: R1 is H and R2 is methyl.

The above-mentioned maytansinoids can be conjugated to anti-CA6 antibodyDS6, or a homologue or fragment thereof, wherein the antibody is linkedto the maytansinoid using the thiol or disulfide functionality that ispresent on the acyl group of an acylated amino acid side chain found atC-3, C-14 hydroxymethyl, C-15 hydroxy or C-20 desmethyl of themaytansinoid, and wherein the acyl group of the acylated amino acid sidechain has its thiol or disulfide functionality located at a carbon atomthat has one or two substituents, said substituents being CH₃, C₂H₅,linear alkyl or alkenyl having from 1 to 10 carbon atoms, branched orcyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical,and in addition one of the substituents can be H, and wherein the acylgroup has a linear chain length of at least three carbon atoms betweenthe carbonyl functionality and the sulfur atom.

A preferred conjugate of the present invention is the one that comprisesthe anti-anti-CA6 antibody DS6, or a homologue or fragment thereof,conjugated to a maytansinoid of formula (VIII):

wherein:Y₁′ represents(CR₇CR₈)_(l)(CR₉═CR₁₀)_(p)(C═C)_(q)A_(r)(CR₅CR₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C═C)_(s)B_(t)(CR₃CR₄)_(n)CR₁R₂S—,wherein:

A, B, and D, each independently is cycloalkyl or cycloalkenyl having3-10 carbon atoms, simple or substituted aryl, or heterocyclic aromaticor heterocycloalkyl radical;

R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₁, and R₁₂ are each independently H, CH₃,C₂H₅, linear alkyl or alkenyl having from 1 to 10 carbon atoms, branchedor cyclic alkyl or alkenyl having from 3 to 10 carbon atoms, phenyl,substituted phenyl or heterocyclic aromatic or heterocycloalkyl radical;and

l, m, n, o, p, q, r, s, and t are each independently 0 or an integer offrom 1 to 5, provided that at least two of l, m, n, o, p, q, r, s and tare non-not zero at any one time.

Preferably, R₁ is H and R₂ is methyl or R₁ and R₂ are methyl.

An even more preferred conjugate of the present invention is the onethat comprises the anti-CA6 antibody DS6, or a homologue or fragmentthereof, conjugated to a maytansinoid of formula (IX-L), (IX-D), or(IX-D,L):

wherein:Y₁ represents (CR₇CR₈)_(l)(CR₅CR₆)_(m)(CR₃CR₄)_(n)CR₁R₂S—,wherein:

R₁ and R₂ are each independently CH₃, C₂H₅, linear alkyl or alkenylhaving from 1 to 10 carbon atoms, branched or cyclic alkyl or alkenylhaving from 3 to 10 carbon atoms, phenyl, substituted phenyl,heterocyclic aromatic or heterocycloalkyl radical, and in addition R₂can be H;

R₃, R₄, R₅, R₆, R₇ and R₈ are each independently H, CH₃, C₂H₅, linearalkyl or alkenyl having from 1 to 10 carbon atoms, branched or cyclicalkyl or alkenyl having from 3 to 10 carbon atoms, phenyl, substitutedphenyl or heterocyclic aromatic or heterocycloalkyl radical;

l, m and n are each independently an integer of from 1 to 5, and inaddition n can be 0; and

May represents a maytansinol which bears the side chain at C-3, C-14hydroxymethyl, C-15 hydroxy or C-20 desmethyl.

Preferred embodiments of formulas (IX-L), (IX-D) and (IX-D,L) includecompounds of formulas (IX-L), (IX-D) and (IX-D,L) wherein:

R₁ is H and R₂ is methyl or R₁ and R₂ are methyl,

R₁ is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H; l and m are each 1;n is 0,

R₁ and R₂ are methyl; R₅, R₆, R₇ and R₈ are each H; l and m are 1; n is0.

Preferably the cytotoxic agent is represented by formula (IX-L).

An further preferred conjugate of the present invention is the one thatcomprises the anti-CA6 antibody DS6, or a homologue or fragment thereof,conjugated to a maytansinoid of formula (X):

wherein the substituents are as defined for formula (IX) above.

Especially preferred are any of the above-described compounds, whereinR₁ is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H, l and m are each 1,and n is 0.

Further especially preferred are any of the above-described compounds,wherein R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ are each H, l and m are 1,and n is 0

Further, the L-aminoacyl stereoisomer is preferred.

Each of the maytansinoids taught in pending U.S. patent application Ser.No. 10/849,136, filed May 20, 2004, may also be used in the cytotoxicconjugate of the present invention. The entire disclosure of U.S. patentapplication Ser. No. 10/849,136 is incorporated herein by reference.

Disulfide-Containing Linking Groups

In order to link the maytansinoid to a cell binding agent, such as theDS6 antibody, the maytansinoid comprises a linking moiety. The linkingmoiety contains a chemical bond that allows for the release of fullyactive maytansinoids at a particular site. Suitable chemical bonds arewell known in the art and include disulfide bonds, acid labile bonds,photolabile bonds, peptidase labile bonds and esterase labile bonds.Preferred are disulfide bonds.

The linking moiety also comprises a reactive chemical group. In apreferred embodiment, the reactive chemical group can be covalentlybound to the maytansinoid via a disulfide bond linking moiety.

Particularly preferred reactive chemical groups are N-succinimidylesters and N-sulfosuccinimidyl esters.

Particularly preferred maytansinoids comprising a linking moiety thatcontains a reactive chemical group are C-3 esters of maytansinol and itsanalogs where the linking moiety contains a disulfide bond and thechemical reactive group comprises a N-succinimidyl orN-sulfosuccinimidyl ester.

Many positions on maytansinoids can serve as the position to chemicallylink the linking moiety. For example, the C-3 position having a hydroxylgroup, the C-14 position modified with hydroxymethyl, the C-15 positionmodified with hydroxy and the C-20 position having a hydroxy group areall expected to be useful. However the C-3 position is preferred and theC-3 position of maytansinol is especially preferred.

While the synthesis of esters of maytansinol having a linking moiety isdescribed in terms of disulfide bond-containing linking moieties, one ofskill in the art will understand that linking moieties with otherchemical bonds (as described above) can also be used with the presentinvention, as can other maytansinoids. Specific examples of otherchemical bonds include acid labile bonds, photolabile bonds, peptidaselabile bonds and esterase labile bonds. The disclosure of U.S. Pat. No.5,208,020, incorporated herein, teaches the production of maytansinoidsbearing such bonds.

The synthesis of maytansinoids and maytansinoid derivatives having adisulfide moiety that bears a reactive group is described in U.S. Pat.Nos. 6,441,163 and 6,333,410, and U.S. application Ser. No. 10/161,651,each of which is herein incorporated by reference.

The reactive group-containing maytansinoids, such as DM1, are reactedwith an antibody, such as the DS6 antibody, to produce cytotoxicconjugates. These conjugates may be purified by HPLC or bygel-filtration.

Several excellent schemes for producing such antibody-maytansinoidconjugates are provided in U.S. Pat. No. 6,333,410, and U.S. applicationSer. Nos. 09/867,598, 10/161,651 and 10/024,290, each of which isincorporated herein in its entirety.

In general, a solution of an antibody in aqueous buffer may be incubatedwith a molar excess of maytansinoids having a disulfide moiety thatbears a reactive group. The reaction mixture can be quenched by additionof excess amine (such as ethanolamine, taurine, etc.). Themaytansinoid-antibody conjugate may then be purified by gel-filtration.

The number of maytansinoid molecules bound per antibody molecule can bedetermined by measuring spectrophotometrically the ratio of theabsorbance at 252 nm and 280 nm. An average of 1-10 maytansinoidmolecules/antibody molecule is preferred.

Conjugates of antibodies with maytansinoid drugs can be evaluated fortheir ability to suppress proliferation of various unwanted cell linesin vitro. For example, cell lines such as the human epidermoid carcinomaline A-431, the human small cell lung cancer cell line SW2, the humanbreast tumor line SKBR3 and the Burkitt's lymphoma line Namalwa caneasily be used for the assessment of cytotoxicity of these compounds.Cells to be evaluated can be exposed to the compounds for 24 hours andthe surviving fractions of cells measured in direct assays by knownmethods. IC₅₀ values can then be calculated from the results of theassays.

PEG-Containing Linking Groups

Maytansinoids may also be linked to cell binding agents using PEGlinking groups, as set forth in U.S. application Ser. No. 10/024,290.These PEG linking groups are soluble both in water and in non-aqueoussolvents, and can be used to join one or more cytotoxic agents to a cellbinding agent. Exemplary PEG linking groups include hetero-bifunctionalPEG linkers that bind to cytotoxic agents and cell binding agents atopposite ends of the linkers through a functional sulfhydryl ordisulfide group at one end, and an active ester at the other end.

As a general example of the synthesis of a cytotoxic conjugate using aPEG linking group, reference is again made to U.S. application Ser. No.10/024,290 for specific details. Synthesis begins with the reaction ofone or more cytotoxic agents bearing a reactive PEG moiety with acell-binding agent, resulting in displacement of the terminal activeester of each reactive PEG moiety by an amino acid residue of the cellbinding agent, to yield a cytotoxic conjugate comprising one or morecytotoxic agents covalently bonded to a cell binding agent through a PEGlinking group.

Taxanes

The cytotoxic agent used in the cytotoxic conjugates according to thepresent invention may also be a taxane or derivative thereof.

Taxanes are a family of compounds that includes paclitaxel (Taxol), acytotoxic natural product, and docetaxel (Taxotere), a semi-syntheticderivative, two compounds that are widely used in the treatment ofcancer. Taxanes are mitotic spindle poisons that inhibit thedepolymerization of tubulin, resulting in cell death. While docetaxeland paclitaxel are useful agents in the treatment of cancer, theirantitumor activity is limited because of their non-specific toxicitytowards normal cells. Further, compounds like paclitaxel and docetaxelthemselves are not sufficiently potent to be used in conjugates of cellbinding agents.

A preferred taxane for use in the preparation of cytotoxic conjugates isthe taxane of formula (XI):

Methods for synthesizing taxanes that may be used in the cytotoxicconjugates of the present invention, along with methods for conjugatingthe taxanes to cell binding agents such as antibodies, are described indetail in U.S. Pat. Nos. 5,416,064, 5,475,092, 6,340,701, 6,372,738 and6,436,931, and in U.S. application Ser. Nos. 10/024,290, 10/144,042,10/207,814, 10/210,112 and 10/369,563.

CC-1065 Analogues

The cytotoxic agent used in the cytotoxic conjugates according to thepresent invention may also be CC-1065 or a derivative thereof.

CC-1065 is a potent anti-tumor antibiotic isolated from the culturebroth of Streptomyces zelensis. CC-1065 is about 1000-fold more potentin vitro than are commonly used anti-cancer drugs, such as doxorubicin,methotrexate and vincristine (B. K. Bhuyan et al., Cancer Res., 42,3532-3537 (1982)). CC-1065 and its analogs are disclosed in U.S. Pat.Nos. 6,372,738, 6,340,701, 5,846,545 and 5,585,499.

The cytotoxic potency of CC-1065 has been correlated with its alkylatingactivity and its DNA-binding or DNA-intercalating activity. These twoactivities reside in separate parts of the molecule. Thus, thealkylating activity is contained in the cyclopropapyrroloindole (CPI)subunit and the DNA-binding activity resides in the two pyrroloindolesubunits.

Although CC-1065 has certain attractive features as a cytotoxic agent,it has limitations in therapeutic use. Administration of CC-1065 to micecaused a delayed hepatotoxicity leading to mortality on day 50 after asingle intravenous dose of 12.5 μg/kg {V. L. Reynolds et al., J.Antibiotics, XXIX, 319-334 (1986)}. This has spurred efforts to developanalogs that do not cause delayed toxicity, and the synthesis of simpleranalogs modeled on CC-1065 has been described {M. A. Warpehoski et al.,J. Med. Chem., 31, 590-603 (1988)}.

In another series of analogs, the CPI moiety was replaced by acyclopropabenzindole (CBI) moiety {D. L. Boger et al., J. Org. Chem.,55, 5823-5833, (1990), D. L. Boger et al., Bio Org. Med. Chem. Lett., 1,115-120 (1991)}. These compounds maintain the high in vitro potency ofthe parental drug, without causing delayed toxicity in mice. LikeCC-1065, these compounds are alkylating agents that bind to the minorgroove of DNA in a covalent manner to cause cell death. However,clinical evaluation of the most promising analogs, Adozelesin andCarzelesin, has led to disappointing results {B.F. Foster et al.,Investigational New Drugs, 13, 321-326 (1996); I. Wolff et al., Clin.Cancer Res., 2, 1717-1723 (1996)}. These drugs display poor therapeuticeffects because of their high systemic toxicity.

The therapeutic efficacy of CC-1065 analogs can be greatly improved bychanging the in vivo distribution through targeted delivery to the tumorsite, resulting in lower toxicity to non-targeted tissues, and thus,lower systemic toxicity. In order to achieve this goal, conjugates ofanalogs and derivatives of CC-1065 with cell-binding agents thatspecifically target tumor cells have been described {U.S. Pat. Nos.5,475,092; 5,585,499; 5,846,545}. These conjugates typically displayhigh target-specific cytotoxicity in vitro, and exceptional anti-tumoractivity in human tumor xenograft models in mice {R. V. J. Chari et al.,Cancer Res., 55, 4079-4084 (1995)}.

Methods for synthesizing CC-1065 analogs that may be used in thecytotoxic conjugates of the present invention, along with methods forconjugating the analogs to cell binding agents such as antibodies, aredescribed in detail in U.S. Pat. Nos. 5,475,092, 5,846,545, 5,585,499,6,534,660 and 6,586,618 and in U.S. application Ser. Nos. 10/116,053 and10/265,452.

Other Drugs

Drugs such as methotrexate, daunorubicin, doxorubicin, vincristine,vinblastine, melphalan, mitomycin C, chlorambucil, calicheamicin,tubulysin and tubulysin analogs, duocarmycin and duocarmycin analogs,dolastatin and dolastatin analogs are also suitable for the preparationof conjugates of the present invention. The drug molecules can also belinked to the antibody molecules through an intermediary carriermolecule such as serum albumin. Doxarubicin and Danorubicin compounds,as described, for example, in U.S. Ser. No. 09/740,991, may also beuseful cytotoxic agents.

Therapeutic Composition

The present invention also provides a therapeutic compositioncomprising:

(a) an effective amount of one or more cytotoxic conjugate, and

(b) a pharmaceutically acceptable carrier.

Similarly, the present invention provides a method for inhibiting thegrowth of selected cell populations comprising contacting target cells,or tissue containing target cells, with an effective amount of acytotoxic conjugate, or therapeutic agent comprising a cytotoxicconjugate, either alone or in combination with other cytotoxic ortherapeutic agents.

The present invention also comprises a method for treating a subjecthaving cancer using the therapeutic composition of the presentinvention.

Cytotoxic conjugates can be evaluated for in vitro potency andspecificity by methods previously described (see, e.g., R. V. J. Chariet al, Cancer Res. 55:4079-4084 (1995)). Anti-tumor activity can beevaluated in human tumor xenograft models in mice by methods alsopreviously described (see, e.g., Liu et al, Proc. Natl. Acad. Sci.93:8618-8623 (1996)).

Suitable pharmaceutically-acceptable carriers are well known and can bedetermined by those of ordinary skill in the art as the clinicalsituation warrants. As used herein, carriers include diluents andexcipients.

Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH ˜7.4, containing or notcontaining about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; andmay also contain an antioxidant such as tryptamine and a stabilizingagent such as Tween 20.

The method for inhibiting the growth of selected cell populations can bepracticed in vitro, in vivo, or ex vivo. As used herein, inhibitinggrowth means slowing the growth of a cell, decreasing cell viability,causing the death of a cell, lysing a cell and inducing cell death,whether over a short or long period of time.

Examples of in vitro uses include treatments of autologous bone marrowprior to their transplant into the same patient in order to killdiseased or malignant cells; treatments of bone marrow prior to itstransplantation in order to kill competent T cells and preventgraft-versus-host-disease (GVHD); treatments of cell cultures in orderto kill all cells except for desired variants that do not express thetarget antigen; or to kill variants that express undesired antigen.

The conditions of non-clinical in vitro use are readily determined byone of ordinary skill in the art.

Examples of clinical ex vivo use are to remove tumor cells or lymphoidcells from bone marrow prior to autologous transplantation in cancertreatment or in treatment of autoimmune disease, or to remove T cellsand other lymphoid cells from autologous or allogeneic bone marrow ortissue prior to transplant in order to prevent graft versus host disease(GVHD). Treatment can be carried out as follows. Bone marrow isharvested from the patient or other individual and then incubated inmedium containing serum to which is added the cytotoxic agent of theinvention. Concentrations range from about 10 μM to 1 pM, for about 30minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation, i.e., the dose, are readilydetermined by one of ordinary skill in the art. After incubation thebone marrow cells are washed with medium containing serum and returnedto the patient by i.v. infusion according to known methods. Incircumstances where the patient receives other treatment such as acourse of ablative chemotherapy or total-body irradiation between thetime of harvest of the marrow and reinfusion of the treated cells, thetreated marrow cells are stored frozen in liquid nitrogen using standardmedical equipment.

For clinical in vivo use, the cytotoxic conjugate of the invention willbe supplied as solutions that are tested for sterility and for endotoxinlevels. Examples of suitable protocols of cytotoxic conjugateadministration are as follows. Conjugates are given weekly for 4 weeksas an i.v. bolus each week. Bolus doses are given in 50 to 100 ml ofnormal saline to which 5 to 10 ml of human serum albumin can be added.Dosages will be 10 μg to 100 mg per administration, i.v. (range of 100ng to 1 mg/kg per day). More preferably, dosages will range from 50 μgto 30 mg. Most preferably, dosages will range from 1 mg to 20 mg. Afterfour weeks of treatment, the patient can continue to receive treatmenton a weekly basis. Specific clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

Examples of medical conditions that can be treated according to the invivo or ex vivo methods of killing selected cell populations includemalignancy of any type including, for example, cancer of the lung,breast, colon, prostate, kidney, pancreas, ovary, cervix and lymphaticorgans, osteosarcoma, synovial carcinoma, a sarcoma or a carcinoma inwhich CA6 is expressed, and other cancers yet to be determined in whichCA6 glycotope is expressed predominantly; autoimmune diseases, such assystemic lupus, rheumatoid arthritis, and multiple sclerosis; graftrejections, such as renal transplant rejection, liver transplantrejection, lung transplant rejection, cardiac transplant rejection, andbone marrow transplant rejection; graft versus host disease; viralinfections, such as mV infection, HIV infection, AIDS, etc.; andparasite infections, such as giardiasis, amoebiasis, schistosomiasis,and others as determined by one of ordinary skill in the art.

Kit

The present invention also includes kits, e.g., comprising a describedcytotoxic conjugate and instructions for the use of the cytotoxicconjugate for killing of particular cell types. The instructions mayinclude directions for using the cytotoxic conjugates in vitro, in vivoor ex vivo.

Typically, the kit will have a compartment containing the cytotoxicconjugate. The cytotoxic conjugate may be in a lyophilized form, liquidform, or other form amendable to being included in a kit. The kit mayalso contain additional elements needed to practice the method describedon the instructions in the kit, such a sterilized solution forreconstituting a lyophilized powder, additional agents for combiningwith the cytotoxic conjugate prior to administering to a patient, andtools that aid in administering the conjugate to a patient.

ADDITIONAL EMBODIMENTS

The present invention further provides for monoclonal antibodies,humanized antibodies and epitope-binding fragments thereof that arefurther labeled for use in research or diagnostic applications. Inpreferred embodiments, the label is a radiolabel, a fluorophore, achromophore, an imaging agent or a metal ion.

A method for diagnosis is also provided in which said labeled antibodiesor epitope-binding fragments thereof are administered to a subjectsuspected of having a cancer, and the distribution of the label withinthe body of the subject is measured or monitored.

EXAMPLES

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention tospecific embodiments.

Example 1 Identification of Antigen Positive and Negative Cell Lines byFlow Cytometry Binding Assays

Flow cytometric analysis was used to localize the DS6 epitope, CA6, tothe cell surface. Human cell lines were obtained from the American TypeCulture Collection (ATCC) with the exception of OVCAR5 (Kearse et al.,Int. J. Cancer 88(6):866-872 (2000)), OVCAR8 and IGROV1 cells (M.Seiden, Massachusetts General Hospital). All cells were grown in RPMI1640 supplemented with 4 mM L-glutamine, 50 U/ml penicillin, 50 μg/mlstreptomycin (Cambrex Bio Science, Rockland, Me.) and 10% v/v fetalbovine serum (Atlas Biologicals, Fort Collins, Colo.), referredhereafter as culture media. Cells were maintained in a 37° C., 5% CO₂humidified incubator.

Cells (1-2×10⁻⁵ cells/well) were incubated, on ice for 3-4 h, withserially diluted concentrations of the DS6 antibody prepared in FACSbuffer (2% goat serum, RPMI) into 96-well plates. The cells were spundown in a table top centrifuge at 1500 rpm for 5 min at 4° C. Afterremoving the media, the wells were then refilled with 150 μl of FACSbuffer. The wash step was then repeated. FITC-labeled goat anti-mouseIgG (Jackson Immunoresearch) was diluted 1:100 to FACS buffer andincubated with the cells for 1 h on ice. The plate was covered in foilto prevent photobleaching of the signal. After two washes, the cellswere fixed with 1% formaldehyde and analyzed on a flow cytometer.

Predominantly, CA6 epitope was found in cell lines of ovarian, breast,cervical, and pancreatic origin (Table 3) as predicted from the tumorimmunohistochemistry. However, some cell lines of other tumor typesexhibited limited CA6 expression. The DS6 antibody binds with anapparent K_(D) of 135.6 pM (in PC-3 cells, Table 3). The maximum meanfluorescence (Table 3) of the binding curves (FIG. 1) in the antigenpositive cell lines are suggestive of the relative antigen density.TABLE 3 Cell Apparent Cell Apparent Line Tissue Antigen MMF* Kd (M) LineTissue Antigen MMF* Kd(M) HL-60 Blood − Caov-3 Ovary + 465.20 5.478 ×10⁻⁰⁹ Jurkat Blood − Caov-4 Ovary + 149.00 4.043 × 10⁻⁰⁹ Namalwa Blood −ES-2 Ovary − U-937 Blood − IGROV1 Ovary − T98G Brain + 35.94 1.775 ×10⁻¹⁰ OV-90 Ovary − BT-20 Breast + 232.20 9.142 × 10⁻¹⁰ OVCAR-3 Ovary −BT-474 Breast − OVCAR5 Ovary + 97.10 1.473 × 10⁻⁰⁹ BT-483 Breast +1911.00 1.366 × 10⁻⁰⁸ OVCAR8 Ovary − BT-549 Breast + 71.39 1.046 × 10⁻⁰⁹PA-1 Ovary − CAMA-1 Breast + 12.46 2.330 × 10⁻⁰⁹ SK-OV-3 Ovary − MCF-7Breast + 81.41 2.890 × 10⁻⁰⁹ SW 626 Ovary − MDA-MB-157 Breast + 8.6351.972 × 10⁻¹⁰ TOV-112D Ovary − MDA-MB-231 Breast + 31.85 1.460 × 10⁻⁰⁹TOV-21G Ovary + 87.79 3.067 × 10⁻¹⁰ MDA-MB-468 Breast + 71.58 8.127 ×10⁻¹⁰ AsPC-1 Pancreas − SK-BR-3 Breast − BxPC-3 Pancreas + 79.99 5.263 ×10⁻⁰⁹ T-47D Breast + 559.58 3.424 × 10⁻⁰⁹ HPAC Pancreas + 2228.00 2.348× 10⁻⁰⁸ ZR-75-1 Breast + 811.67 4.299 × 10⁻⁰⁹ HPAF-II Pancreas + 266.502.811 × 10⁻⁰⁹ HeLa Cervix + 242.50 6.938 × 10⁻¹⁰ Hs766T Pancreas +182.90 2.319 × 10⁻⁰⁹ KB Cervix + 119.56 1.110 × 10⁻⁰⁹ MIAPaCa2 Pancreas− WISH Cervix + 1133.55 2.380 × 10⁻⁰⁹ MPanc96 Pancreas − Colo205 Colon −SU.86.86 Pancreas + 36.86 1.043 × 10⁻⁰⁹ DLD-1 Colon − SW1990 Pancreas +36.17 3.679 × 10⁻¹⁰ HCT-8 Colon − PC-3 Prostate + 24.81 1.356 × 10⁻¹⁰HT-29 Colon − A375 Skin − Caki-1 Kidney − SKMEL28 Skin − A549 Lung − KLEUterus − SW2 Lung −*average maximum relative mean fluorescence

Example 2 Characterization of DS6 Epitope

The properties of the DS6 antigen, CA6, were analyzed by immunoblottingthe dot blots of CA6-positive cell lysates (Caov-3) that were digestedwith proteolytic (pronase and proteinase K) and/or glycolytic(neuraminidase and periodic acid) treatments. For positive controls,other antibodies recognizing a variety of epitope types were tested onlysates of antigen positive cell lines (Caov-3 and CM1; Colo205 andC242; SKMEL28 and R₂₄). CM1 is an antibody recognizing a protein epitopeof the variable number tandem repeat domain (VNTR) of Muc-1 and thus,provides a control for a protein epitope. C242 binds to a novelcolorectal cancer specific sialic acid-dependent glycotope on Muc-1(CanAg) which provides a control for a glycotope on a protein. R24 bindsto the GD3 ganglioside that is specific for melanoma and thus provides acontrol for a glycotope on a non-protein scaffold.

Caov-3, Colo205, and SKMEL28 cells were plated in 15 cm tissue cultureplates. Culture media (30 mL/plate) was refreshed the day before lysis.A modified RIPA buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM EDTA,1% NP40, 0.25% sodium deoxycholate), protease inhibitors (PMSF,Pepstatin A, Leupeptin, and Aprotinin), and PBS were pre-chilled on ice.After the culture media was aspirated from the plates, the cells werewashed twice with 10 ml of chilled PBS. All of the subsequent steps wereconducted on ice and/or in a 4° C. cold room. After the last wash of PBSwas aspirated, the cells were lysed in 1-2 mL of lysis buffer (RIPAbuffer with freshly added protease inhibitors to a final concentrationof 1 mM PMSF, 1 μM Pepstatin A, 10 μg/ml Leupeptin, and 2 μg/mlAprotinin). The lysates were scraped off of the plates using a celllifter and triturated by pipetting the suspensions up and down (5-10times) with an 18G needle. The lysates were rotated for 10 min and thencentrifuged in a microcentrifuge at maximum (13 K rpm) for 10 min. Thepellets were discarded and the supernatants were then assayed using aBradford Protein assay kit (Biorad).

The lysates (2 μl) were pipetted directly onto dry 0.2 μm nitrocellulosemembranes. The spots were allowed to air dry for approximately 30 min.The membrane was sectioned into pieces that each contained a singlespot. Spots were incubated in the presence of pronase (1 mg/ml enzyme,50 mM Tris pH 7.5, 5 mM CaCl₂), proteinase K (1 mg/ml enzyme, 50 mM TrispH 7.5, 5 mM CaCl₂), neuraminidase (20 mU/ml enzyme, 50 mM sodiumacetate pH 5, 5 mM CaCl₂, 100 μg/ml BSA) or periodic acid (20 mM, 0.5Msodium acetate pH 5) for 1 h at 37° C. Reagents were purchased fromRoche (enzymes) and VWR (periodic acid). Membranes were washed (5 min)in T-TBS wash buffer (0.1% Tween 20, 1×TBS), blocked in blocking buffer(3% BSA, T-TBS) for 2 h at room temperature, and incubated overnightwith 2 μg/ml of primary antibody (i.e. DS6, CM1, C242, R24) in blockingbuffer at 4° C. The membranes were washed three times for 5 min in T-TBSand then incubated in HRP-conjugated goat anti-mouse (or human) IgGsecondary antibody (Jackson Immunoresearch; 1:2000 dilution in blockingbuffer) for 1 h at room temperature. The immunoblots were washed threetimes and developed using an ECL system (Amersham).

The immunoblots (FIG. 2) of the digested control lysates showed that theCM1 signal was destroyed by the proteolytic treatments while the signalsof the glycolytic digests were unaffected as would be expected for anantibody recognizing a protein epitope. The C242 signal was destroyed byeither the proteolytic or glycolytic treatments as would be expected foran antibody recognizing a glycotope found on a protein. The R24 signal,unaffected by the proteolytic treatments, was abolished withneuraminidase or periodate treatments as expected for an antibodyrecognizing a ganglioside. The DS6 immunoblot of the digested Caov-3lysate dot blots showed signal loss upon treatment with either theproteolytic and glycolytic compounds. Thus, like C242, DS6 binds to acarbohydrate epitope on a proteinaceous core. Furthermore, the signal inthe DS6 immunoblot was sensitive to neuraminidase treatment. Therefore,CA6, like CanAg, is a sialic acid-dependent glycotope.

In order to confirm the carbohydrate nature of CA6, Caov-3 lysate wasspotted onto PVDF membrane and treated with the chemical deglycosylatingagent, trifluoromethane sulfonic acid (TFMSA), under nitrogen at ambienttemperature for 5 minutes. The blot was washed with T-TBS andimmunoblotted with either CM1 or DS6 (FIG. 3). The DS6 signal wasdestroyed upon the acid treatment providing further evidence that CA6 isa glycotope. The enhancement of the CM1 signal upon TFSMA treatmentindicates that the acid treatment did not affect the protein on thefilter and suggests that the glycolytic treatment unmasked the proteinepitope recognized by CM1.

To further elucidate the structure of the carbohydrate on which CA6resides, dot blots were digested with N-glycanase, O-glycanase, and/orsialidase (FIG. 4). Caov-3 cell lysates (100 μg, 30 μl) were incubatedat 100° C. for 5 min with 2.5 μl of denaturation buffer (Glyko)containing SDS and β-mercaptoethanol. The denatured lysates were thendigested with 1 μl of N-glycanase, O-glycanase, and/or Sialidase A(Glyko) at 37° C. for 1 h. The digested lysates were then spotted (2 μl)onto nitrocellulose and immunoblotted as described above.

N-glycanase had no apparent effect on the DS6 immunoblot signals.However, samples digested with sialidase produced no signal. BecauseO-glycanase cannot digest sialyated O-linked carbohydrates withoutpretreatment with sialidase, the DS6 signal of samples processed withO-glycanase alone would not be affected. N-glycanase, in contrast, doesnot require pretreatment with any glycosidic enzymes for activity. Thefact that N-glycanase treatment does not affect the DS6 signal suggeststhat the CA6 epitope is most likely present on sialyated O-linkedcarbohydrate chains.

Example 3 Elucidation of the Antigen on which the CA6 Epitope is Found

To identify the antigen on which the CA6 sialoglycotope is found, DS6immunoprecipitates were analyzed by SDS-PAGE and Western blotting. Celllysate supernatants (1 mL/sample; 3-5 mg protein) were pre-cleared withProtein G beads (30 μl), equilibrated with 1 ml of RIPA buffer, for 1-2h, with rotation, at 4° C. All of the subsequent steps were conducted onice and/or in a 4° C. cold room. The pre-cleared beads were spun downbriefly (2-3 s) in a microcentrifuge. The pre-cleared supernatants weretransferred to fresh tubes and incubated overnight with 2 μg of DS6,with rotation. Fresh, equilibrated Protein G beads (30 μl) were added tothe lysates and incubated for 1 h, with rotation. The bead-lysatesuspensions were briefly spun down in a microcentrifuge and samples ofthe post-immunoprecipitation lysates were optionally taken. The beadswere washed 5-10 times with 1 mL of RIPA buffer.

Immunoprecipitated DS6 samples were then digested with 30 μlneuraminidase (20 mU neuraminidase (Roche), 50 mM sodium acetate pH 5, 5mM CaCl₂, 100 μg/ml BSA) or 30 μl periodic acid (20 mM periodic acid(VWR), 0.5M sodium acetate pH 5) for 1 h at 37° C. They were thenresuspended in 30 μl of 2× sample loading buffer (containingα-mercaptoethanol). The beads were boiled for 5 min and the loadingbuffer supernatants were loaded onto 4-12% or 4-20% Tris-Glycine gels(Invitrogen). The gels were run in Laemmli electrophoresis runningbuffer at 125 V for 1.5 h. The gel samples were transferred, overnightat 20 mA, onto 0.2 μm nitrocellulose membranes (Invitrogen) using a MiniTrans-blot transfer apparatus (Biorad). Membranes were immunoblottedwith DS6 as described above in Example 2.

Alternatively, the immunoprecipitated beads were first denatured andthen enzymatically digested with N-glycanase, O-glycanase and/orsialidase A (Glyko). The beads were resuspended in 27 μl incubationbuffer and 2 μl denaturation solution (Glyko) and incubated at 100° C.for 5 minutes. After cooling to room temperature, detergent solution (2μl) was added and the samples were incubated with 1 μL of N-glycanase,O-glycanase, and/or Sialidase A at 37° C. for 4 h. After adding 5×sample loading buffer (7 μl), the samples were boiled for 5 min. Thesamples were subjected to SDS-PAGE and immunoblotted as described above.

DS6 immunoprecipitates a>250 kDa protein band that can be seen inantigen positive cell lysates (FIGS. 5A, B, and C). In some cell lines(i.e. T-47D), a doublet is observed. The >250 kDa band was abolished inCaov-3 immunoprecipitates that were treated with neuraminidase orperiodic acid (FIGS. 5 A and B) suggesting that the CA6 epitope resideson the >250 kDa band. The >250 kDa band was also shown to be insensitiveto N-glycanase treatment of immunoprecipitates consistent with CA6residing on an O-linked carbohydrate (FIG. 5F). Further supporting thatthe 250 kDa band is the CA6 antigen is the fact that DS6immunoprecipitates no such band from DS6 antigen negative cells (FIGS.5D and E).

Several lines of evidence suggested that the CA6 antigen was Muc1.Because of the high molecular weight and the sensitivity to O-linkedcarbohydrate-specific glycolytic enzymes, it seemed likely that the CA6antigen was a mucin. Mucin overexpression is well characterized intumors particularly of the breast and ovary, consistent with the majortumor reactivities of DS6. Furthermore, CA6, like CanAg (asialoglycotope on Muc1), is not susceptible to perchloric acidprecipitation suggesting the CA6 antigen is heavily O-glycosylated. Theobservation that in some DS6 expressing cell lines, DS6immunoprecipitated a doublet of >250 kDa suggested that the CA6 wasMuc1. A hallmark of Muc1 in humans is the presence of two distinct Muc1alleles differing in number of tandem repeats resulting in theexpression of two Muc1 proteins of different molecular weights.

To test whether CA6 is found on Muc1, DS6 immunoprecipitates from Caov-3lysate were subjected to SDS-PAGE and immunoblotted with either DS6 or aMuc1 VNTR antibody, CM1. As can be seen in FIG. 6A, CM1 reacts stronglywith the >250 kDa band immunoprecipitated by DS6. In FIG. 6B, DS6 andCM1 immunoprecipitates from HeLa cell lysate show the same >250 kDadoublet when immunoblotted with either DS6 or CM1. These resultsindicate that the CA6 epitope is indeed located on the Muc-1 protein.The DS6 doublet seen in HeLa (and T-47D) cells can be explained by thefact that Muc-1 expression is directed by distinct alleles havingdiffering number of tandem repeats.

Although CM1 and DS6 bind to the same Muc-1 protein, they are distinctepitopes. Chemical deglycosylation of Caov-3 lysate dot blots bytrifluoromethane sulfonic acid (TFMSA) abolished the DS6 signal (FIG.3). However, this same treatment enhanced the CM1 signal.Deglycosylation may have revealed hidden epitopes for the CM1 antibody.Furthermore, a comparison of the flow cytometry binding results of DS6and CM1 (Table 4) demonstrates that the CA6 epitope does not exist onevery cell expressing Muc1. It is interesting to note that the CA6epitope is not expressed on Colo205 (Table 3), a cell line known toexpress high levels of the Muc1 CanAg sialoglycotope. TABLE 4 DS6 CM1Cell Apparent Apparent Line MMF* Kd (M) MMF* Kd (M) DS6 positive & BT54971.39 1.046 × 10⁻⁰⁹ 187.90 6.056 × 10⁻⁰⁹ CM1 positive CaOV3 465.20 5.478× 10⁻⁰⁹ 1031.00 7.479 × 10⁻⁰⁹ HeLa 242.50 6.938 × 10⁻¹⁰ 334.80 2.907 ×10⁻⁰⁹ KB 119.56 1.110 × 10⁻⁰⁹ 338.00 5.345 × 10⁻⁰⁹ MCF7 81.41 2.890 ×10⁻⁰⁹ 1023.00 8.694 × 10⁻⁰⁹ DS6 negative & KLE 27.48 — 561.70 8.156 ×10⁻⁰⁹ CM1 positive OVCAR3 21.19 — 192.50 5.949 × 10⁻⁰⁹ SKOV3 17.53 —49.41 6.246 × 10⁻⁰⁹*MMF = maximum mean relative fluorescence

Example 4 Quantitative Analysis of Shed CA6 Epitope

Because the CA6 epitope resides on Muc1, a molecule known to be shedinto the blood stream in many cancer patients, a quantitative approachwas undertaken in order to determine whether such levels would beprohibitive for DS6 antibody therapy. Binding of circulating antibody toantigen is thought to lead to rapid clearance of immune complexes fromthe blood. If a significant portion of the administered antibody dose israpidly removed from circulation the amount reaching the tumor is likelyto be diminished resulting in decreased anti-tumor activity of anantibody therapeutic. When the antibody is conjugated to a highly potentcytotoxic compound the rapid clearance of conjugate could potentiallyincrease non-specific toxicity. Thus, in the case of antibody-small drugconjugates such as DS6-DM1, high levels of shed antigen might beexpected to both reduce the anti-tumor effect and increase thedose-limiting toxicity.

Recent clinical trials of antibody therapeutics have yielded informationas to the impact of shed antigen concentration on pharmacokinetics. Forexample, in clinical trials with trastuzumab (Herceptin), an antibodyused for the treatment of her2/neu-expressing metastatic breast cancer,the pharmacokinetics of trastuzumab clearance was shown to be unalteredwhen the shed Her2/neu level was less than 500 ng/mL (Pegram et al., J.Clin. Oncol. 16(8):2659-71 (1998). Assuming a molecular weight of shedHer2/neu of 110,000 Daltons, a molar concentration shed Her2/neu below4.5 nM appears to have little influence on the pharmacokinetics.

In another example, a clinical trial with cantuzumab mertansine(huC242-DM1) indicated that there was no correlation with pretreatmentshed CanAg (C242 epitope) levels and pharmacokinetics of antibodyclearance (Tolcher et al., J. Clin. Oncol. 21(2):211-22 (2003). TheCanAg epitope, similar to the CA6 epitope recognized by DS6, is a uniquetumor-specific O-linked sialoglycotope on Muc1. However, theheterogeneous nature of the CanAg epitope makes it difficult to quantifyin molar terms. In the general population Muc1 alleles vary in lengthdepending upon the number of tandem repeats in the variable numbertandem repeat (VNTR) domain. Several sites for O-linked glycosylationoccur in each tandem repeat. Adding to the complexity of CanAgexpression is the cell-to-cell variation in inherent glycosyltransferase activity. Thus a wide range of CanAg epitopes per Muc1molecule are possible even in a single patient. Moreover, the ratio ofCanAg epitope per Muc1 molecule will be different across a population ofpatients. For this reason, shed CanAg in serum samples is measured bysandwich ELISA where shed Muc1 with CanAg epitope is captured by C242and detected by a biotinylated C242/Streptavidin HRP system. The shedCanAg is quantified in standardized units (U) proportional to the numberof epitopes per ml of serum rather than by a molar concentration ofMuc1. By analogy, a similar situation occurs for the quantification ofshed CA6 epitopes. In contrast, for trastuzumab there is only oneepitope per shed her2/neu molecule vastly simplifying the quantificationof shed antigen.

In order to relate CA6 shed epitope levels to those found in clinicaltrials with trastuzumab and cantuzumab mertansine, a method forobtaining molar concentrations of complex shed epitopes such assialoglycotopes on Muc1 was developed. First, a simple sandwich ELISAassay for DS6 was established. A representation of the assay is shown inFIG. 7A. DS6 was used to capture Muc1 having CA6 epitope. Because eachMuc1 molecule has multiple CA6 epitopes, biotinylated DS6 was also usedas the tracer antibody. Biotinylated DS6 bound to captured CA6 wasdetected by Streptavidin-HRP using ABTS as the substrate. CA6 epitopewas captured from ovarian cancer patient serum or from standards whichcome from a commercially available Muc1 test kit (CA15-3) used tomonitor shed Muc1 in breast cancer patients. DS6 units/ml werearbitrarily set equal to CA15-3 standards units/ml.

In FIG. 7B is shown the results of the DS6 sandwich ELISA in whichCA15-3 standards were used. The curve generated is very similar to thatobtained with CA15-3 standards in the CA15-3 assay. In order to convertDS6 unit/ml to a molar concentration of CA6 a standard curve forbiotinylated DS6 which converts signal to picograms of DS6 is required.Assuming a one-to-one stoichiometry between CA6 epitope and biotinylatedDS6 antibody and a molecular weight of 160,000 Daltons for biotinylatedDS6 the moles of CA6 captured per volume of sample added can becomputed.

In FIGS. 8A and B are representations of two alternative means ofgenerating a standard curve for biotinylated DS6. In FIG. 8A, Goatanti-mouse IgG polyclonal antibody is used to capture biotinylated DS6which is in turn detected in a manner identical to that used in thesandwich ELISA assay shown in FIG. 7. In the method shown in FIG. 8Bbiotinylated DS6 is plated directly onto the ELISA plate and detected asin FIG. 8A. As seen in FIG. 8C the biotinylated DS6 standard curvesgenerated by each method are in good agreement.

In Table 5 the analysis of ovarian cancer patient serum samples forvarious shed antigens is shown. CA125 ELISA is generally used to monitorthe treatment of ovarian cancer patients by measuring shed CA125units/ml. The CA125 status was provided with the serum samples. CA15-3ELISA is generally used to monitor the treatment of breast cancerpatients by measuring the units/ml of shed Muc1 using capture anddetections antibodies recognizing epitopes distinct from that recognizedby DS6. In Table 5, CA15-3 is measured in ovarian cancer patients serumsamples. TABLE 5 Serum CA125¹ CA15-3¹ DS6² DS6³ DS6⁴ No. (U/ml) (U/ml)(U/ml) (pM) (pM) 4 72.80 117.72 29.79 52.13 188.94 5 3651.90 98.19567.02 654.44 >2560.00 6 930.50 87.08 504.15 667.56 2505.00 7 76.0072.70 135.65 246.94 778.25 8 32.50 18.44 39.96 65.19 239.88 9 551.70292.39 >975.61 1512.31 >2560.00 10 90.00 42.40 49.48 85.19 305.88 11200.50 60.58 92.32 152.38 526.75 12 283.00 35.67 83.65 135.81 485.06 13197.50 20.61 35.92 61.06 216.25 14 100.60 6.13 12.39 23.19 88.06 1534.60 59.18 199.85 286.63 1228.56 17 196.40 56.75 66.53 130.44 405.88 1816.90 30.45 34.43 60.81 223.69 19 22.00 263.93 118.98 191.69 728.94 22110.70 21.44 16.46 29.94 111.38¹determined by commercial ELISA kit²determined by commercial CA15-3 standards (1 CA15-3 U = 1 DS6 U)³goat anti-mouse IgG & biotin-DS6 standard curve⁴biotin-DS6 standard curve

For the CA15-3 values reported in Table 5, a commercially availableCA15-3 Enzyme Immuno Assay kit from CanAg Diagnostics was used. For theDS6 units/ml a standard curve was generated using the CA15-3 standards(from the CA15-3 Enzyme Immuno Assay kit from CanAg Diagnostics) in theDS6 sandwich ELISA. DS6 units/ml were arbitrarily set equal to CA15-3units/ml. In the last two columns picomolar (pM) shed CA6 was calculatedusing the biotinylated DS6 standard curves shown in FIG. 8C.

For the quantitative analysis of CanAg levels, CanAg serum levels werethose reported for patients participating in a cantuzumab mertansineclinical trial prior to treatment (Tolcher et al., J. Clin. Oncol.21(2):211-22 (2003). An ELISA assay analogous to the one described forDS6 was used to make a CanAg standard curve using CanAg standards. C242was used to capture the CanAg standards. Detection of captured CanAg wasachieved using biotinylated C242 tracer followed by development withstreptavidin-HRP using ABTS as substrate. A biotinylated-C242 standardcurve was constructed as done for biotinylated DS6 allowing for theconversion of units/ml to a molar concentration of circulating CanAgepitopes. In Table 6 CanAg levels from cantuzumab mertansine clinicaltrial patients are reported along with the corresponding calculatedmolar concentrations of circulating CanAg. TABLE 6 CanAg¹ CanAg² CanAg³(U/ml) (pM) (pM) 31240 19185.7 34592.8 8687 3535 9619.3 7456 4579 8256.23686 2263.7 4081.6 1447 888.7 1602.3 1262 775 1397.4 718 441 795.1 547335.9 605.7 394 242 436.3 381 234 421.9 329 202.1 364.3 322 197.8 356.6306 187 338.8 284 174.4 314.5 247 151.7 273.5 242 148.6 268 229 140.6253.6 227 139.4 251.4 184 113 203.7 120 73.7 132.9 107 65.7 118.5 10061.4 110.7 81 49.7 89.7 81 49.7 89.7 67 41.1 74.2 53 32.5 58.7 45 27.649.8 43 26.4 47.6 39 24 43.2 36 22.1 39.9 24 14.7 26.6 18 11.1 19.9 1710.4 18.8 <10 6.1 11.3 <10 6.1 11.3 <10 6.1 11.3 <10 6.1 11.3¹pretreatment levels of circulating CanAg measured by sandwich ELISA²goat anti-mouse IgG & biotin-C242 standard curve³biotin-C242 standard curve

A comparison of the pM levels of shed CA6 in ovarian cancer patientswith those calculated for shed CanAg in CanAg-positive cancer patientsshows that in general shed CA6 levels are similar to shed CanAg levels.Furthermore, only 2 out of 16 ovarian cancer patients serum samplespotentially have CA6 levels greater than 4.5 nM, (serum samples 5 and 9for which the signal was out of the range of the standard curve), thelevel above which altered herceptin pharmacokinetics was observed inclinical trials with Her2/neu-positive breast cancer patients. CanAglevels above 4.5 nM were only seen in 3 out of 37 clinical trialpatients. In this clinical trial there was no correlation with shedCanAg levels and more rapid clearance of cantuzumab mertansine. However,the patient with the highest CanAg level (31240 U/ml) was only sampledfor 8 hours post-transfusion. These results indicate that certainepitopes of Muc1, such as CA6 and CanAg, while shed in cancer patients,are not shed at levels prohibitive for antibody therapeutic treatment.

Example 6 Cloning Murine DS6 Antibody Variable Regions

Murine monoclonal antibodies such as DS6 have limited utility in aclinical setting because they are recognized as foreign by the humanimmune system. Patients quickly develop human anti-mouse antibodies(HAMA) resulting in rapid clearance of murine antibodies. For thisreason, the variable region of murine DS6 (muDS6) was resurfaced toproduce humanized DS6 (huDS6) antibodies.

The murine DS6 antibody variable regions were cloned by RT-PCR. TotalRNA was purified from a confluent T175 flask of DS6 hybridoma cellsusing the Qiagen RNeasy miniprep kit. RNA concentrations were determinedby UV spectrophotometry and RT reactions were done with 4-5 μg total RNAusing the Gibco Superscript II kit and random hexamer primers.

PCR reactions were performed with degenerate primers based on thosedescribed in Wang Z et al., J Immunol Methods. January 13;233(1-2):167-77 (2000). The RT reaction mix was used directly fordegenerate PCR reactions. The 3′ light chain primer, HindKL,

(TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC)

(SEQ ID NO:25)

and 3′ heavy chain primer, BamIgG1,

(GGAGGATCCATAGACAGATGGGGGTGTCGTTTTGGC) (SEQ ID NO:26)

were used, and for the 5′ end PCR primers were Sac1 MK

(GGGAGCTCGAYATTGTGMTSACMCARWCTMCA) (SEQ ID NO:27) for the light chainand an equal mix of EcoR1MH1

(CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC) (SEQ ID NO:28) and EcoR1MH2(CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG) (SEQ ID NO:29) for the heavy chain(mixed bases: H=A+T+C, S=G+C, Y=C+T, K=G+T, M=A+C, R=A+G, W=A+T,V=A+C+G, N=A+T+G+C).

PCR reactions were standard except they were supplemented with 10% DMSO(50 μl reaction mixes contained final concentrations of 1× reactionbuffer (ROCHE), 2 mM each dNTP, 1 mM each primer, 2 μl RT reaction, 51μl DMSO, and 0.5 μl Taq (ROCHE)). The PCR reactions were performed on anMJ research thermocycler using a program adapted from Wang Z et al., (JImmunol Methods. January 13; 233(1-2):167-77 (2000)): 1) 94° C., 3 min;2) 94° C., 15 sec; 3) 45° C., 1 min; 4) 72° C., 2 min; 5) cycle back tostep #2 29 times; 6) finish with a final extension step at 72° C. for 10min. The PCR products were cloned into pBluescript II SK+ (Stratagene)using restriction enzymes created by the PCR primers. Seqwrightsequencing services sequenced the heavy and light chain clones.

In order to confirm the 5′ end cDNA sequences, additional PCR andcloning was done. The DS6 light chain and heavy chain cDNA sequences,determined from the degenerate PCR clones, were plugged into the NCBI'sBlast search website and murine antibody sequences with signal sequencesubmitted were saved. PCR primers were designed from these signalpeptides using conserved stretches among the related DNA sequences.EcoRI restriction sites were added to the leader sequence primers (Table7) and these were used in RT-PCR reactions as described above. TABLE 7DS6 Signal Sequence Degenerate Primers Name Sequence Heavy Chain -DS6HClead ttttgaattcaataactacaggtgtccact - SEQ ID NO: 30 Light Chain -KTILClead ttttgagctccagattttcagcttcctgct - SEQ ID NO: 31

Several individual light and heavy chain clones were sequenced toidentify and avoid possible polymerase generated sequence errors. Only asingle sequence was obtained for both the light chain and heavy chainRT-PCR clones. These sequences were sufficient to design primers thatcould amplify the murine DS6 light and heavy chain sequences extendinginto the signal sequence. The subsequent clones from these follow-up PCRreactions confirmed the 5′ end sequences of the variable region that hadbeen altered by the original degenerate primers. The cumulative resultsfrom the various cDNA clones provided the final murine DS6 light andheavy chain sequences presented in FIG. 9. Using Kabat and AbMdefinitions, the three light chain and heavy chain CDRs were identified(FIGS. 9 and 10). A search of the NCBI IgBlast database indicates thatthe muDS6 antibody light chain variable region most likely derives fromthe murine IgV_(κ) ap4 germline gene while the heavy chain variableregion most likely derives from the murine IgVh J558.41 germline gene(FIG. 11).

Example 7 Determination of the Variable Region Surface Residues of DS6Antibody

The antibody resurfacing techniques described by Pedersen et al. (1994)and Roguska et al. (1996) begin by predicting the surface residues ofthe murine antibody variable sequences. A surface residue is defined asan amino acid that has at least 30% of its total surface area accessibleto a water molecule. In the absence of a solved structure to find thesurface residues for muDS6, we aligned the ten antibodies with the mosthomologous sequences in the set of 127 antibody structure files (FIG.12). The solvent accessibility for each Kabat position was averaged forthese aligned sequences (FIGS. 13A and B).

Surface positions with average accessibilities of between 25% and 35%were subjected to a second round of analysis by comparing a subset ofantibodies containing two identical residues flanking on either side(FIGS. 13A and B). After the second round analysis, the 21 predictedsurface residues for the muDS6 heavy chain were increased to 23, addingTyr3 and Lys23 to the list of residues with predicted surfaceaccessibility greater than 30%. In most of our resurfaced antibodies theKabat definition of the heavy chain CDR1 is used, but for DS6 the AbMdefinition was inadvertently used during the calculations so the heavychain residue T28 was not defined as a framework surface residue as itmight otherwise have been. The number of light chain surface positionswas reduced from 16 to 15 because the predicted surface accessibility ofAla80 was reduced from 30.5% to 27.8% in the second round analysis.Together, the muDS6 heavy and light chain variable sequences have 38predicted surface accessible framework residues.

Example 8 Human Antibody Selection

The surface positions of the murine DS6 variable region were compared tothe corresponding positions in human antibody sequences in the Kabatdatabase (Johnson G, Wu T T. Nucleic Acids Res. January 1; 29(1):205-6(2001)). The antibody database management software SR (Searle, 1998) wasused to extract and align the surface residues from natural heavy andlight chain human antibody pairs. The human antibody variable regionsurface with the most identical surface residues, with specialconsideration given to positions that come within 5 Å of a CDR, waschosen to replace the murine DS6 antibody variable region surfaceresidues.

Example 9 Expression Vector for Chimeric and Humanized Antibodies

The light and heavy chain paired sequences were cloned into a singlemammalian expression vector. The PCR primers for the human variablesequences created restriction sites that allowed the human signalsequence to be added in the pBluescriptII cloning vector. The variablesequences could then be cloned into the mammalian expression plasmidwith EcoRI and BsiWI or HindIII and ApaI for the light chain or heavychain respectively (FIG. 14). The light chain variable sequences werecloned in-frame onto the human IgKappa constant region and the heavychain variable sequences were cloned into the human IgGamma1 constantregion sequence. In the final expression plasmids, human CMV promotersdrive the expression of both the light and heavy chain cDNA sequences.

Example 10 Identification of Residues that may Negatively Affect DS6Activity

In most of the humanizations to date a molecular model of the subjectantibody has been built to identify residues proximal to a CDR aspotential problem residues. With an expanding number of resurfacedantibodies to work from, historical experience is at least as effectiveat predicting problems as building a model, so no molecular model wasbuilt for DS6. Instead, the murine DS6 surface residues were comparedwith those of previously resurfaced antibodies and residues with low tohigh risk for affecting the antibody's binding activity were identified.

Similar sets of residues are repeatedly identified as being within 5 Åof a CDR in both the available solved antibody structures and themolecular models from previous humanizations. Using this data, Table 1gives the murine DS6 residues that are likely proximal to and possiblywithin 5 Å of a CDR. Many of these positions have also been changed inprevious humanizations, but only heavy chain position 74 has everresulted in a loss of binding activity. The murine residue was retainedin this position in both huC242 and huB4 in order to conserve thebinding activity of the murine antibody. On the other hand, this sameposition was changed to the corresponding human residue in humanized6.2G5C6 without loss of activity (6.2G5C6 is the anti-IGF1-R antibodyoften referred to simply as anti-C6). While any of the residues in Table1 could present a problem in the humanized antibody, the heavy chainresidue P73 will be of particular concern due to previous experiences inthis position.

Example 11 Selection of the Most Homologous Human Surface

Candidate human antibody surfaces for resurfacing muDS6 were pulled fromthe Kabat antibody sequence database using SR software. This softwareprovides an interface to search only specified residue positions againstthe antibody database. To preserve the natural pairs, the surfaceresidues of both the light and heavy chains were compared together. Themost homologous human surfaces from the Kabat database were aligned inorder of rank of sequence identity. The top 3 surfaces as aligned by theSR Kabat database software are given in Table 2. The surfaces were thencompared to identify which human surfaces would require the leastchanges to the residues identified in Table 1. The anti-Rh(D) antibody,28E4 (Boucher et al, 1997), requires the least number of surface residuechanges (11 total) and only 3 of these residues are included in the listof potential problem residues. Since the 28E4 antibody provides the mosthomologous human surface, it is the best candidate to resurface muDS6.

Example 12 Construction of the DNA Sequences for Humanized DS6Antibodies

The 11 surface residue changes for DS6 were made using PCR mutagenesis.PCR mutagenesis was performed on the murine DS6 variable region cDNAclones to build the resurfaced, human DS6 gene. Humanization primer setswere designed to make the amino acid changes required for resurfacedDS6, shown below in Table 8. TABLE 8 Primer Name Primer SequenceDS6HCapa cgatgggcccttggtggaggctgcagagacagtgaccaga SEQ ID NO: 32 DS6LCBsittttcgtacgtttcagctccagcttggt SEQ ID NO: 33 DS6HC5endcaggtgtacactcccaggcttatctccagcagtct SEQ ID NO: 34 huC6HCApacgatgggcccttggtggaggcggcagagacagtgaccaga SEQ ID NO: 35 ds61c5etcaggtgtacactccgagattgttctcacccagtctccagcaacc SEQ ID NO: 36 atgtctgcatctds6LCr18 ggcactgcaggttatggtgaccctctcccctggaga SEQ ID NO: 37 ds61cs77fcaatcagcagcatggaggctgaaga SEQ ID NO: 38 ds61cs77rgcctccatgctgctgattgtgaga SEQ ID NO: 39 DS6HCvvkpcaggtgtacactcccaggctcagctcgtgcagtctggggctgag SEQ ID NO: 40gtggtgaagcccggggcctcagt DS6HCt ttgactgcagacacatcctccagcaca SEQ ID NO: 41ds6hcQT gtgtctgcagtcaatgtggccttgccctggaacttctgat SEQ ID NO: 42huDS6HCapa cgatgggcccttggtggaggcggcagagacagtgacaaga SEQ ID NO: 43

PCR reactions were standard except they were supplemented with 10% DMSO(50 μl reaction mixes contained final concentrations of 1× reactionbuffer (ROCHE), 2 mM each dNTP, 1 mM each primer, 100 ng template, 5 μlDMSO, and 0.5 μl Taq (ROCHE)). They were run on an MJ Researchthermocycler with the following program: 1) 94° C., 1 min; 2) 94° C., 15sec; 3) 55° C., 1 min; 4) 72° C., 1 min; 5) cycle back to step #2 29times; 6) finish with a final extension step at 72° C. for 4 min. ThePCR products were digested with their corresponding restriction enzymesand cloned into the pBluescript cloning vectors. Clones were sequencedto confirm the amino acid changes.

Since changing heavy chain residue P73 has caused problems in the past,two versions of the heavy chain were built, one with the human 28E4 T73and one retaining the murine P73. The other 10 surface residue werechanged from murine to the human 28E4 residue in both versions ofhumanized DS6 (Table 2). Sticking with the usual naming method, the mosthuman is version 1.0 since it has all 11 human surface residues. Theheavy chain version retaining the murine P73 is named version 1.2 incase further versions are required so version 1.1 is reserved for aversion containing the maximum number of murine residues. The amino acidsequences of the two humanized versions are shown aligned with themurine DS6 amino acid sequence in FIGS. 15A and B. Both of the humanizedDS6 antibody genes were cloned into the antibody expression plasmid(FIG. 14) for transient and stable transfections. The cDNA and aminoacid sequences of the humanized versions 1.01 and 1.21 are light chainvariable region are the same and shown in FIG. 16. The heavy chain cDNAand amino acid sequences of the humanized versions 1.01 and 1.21 areshown in FIGS. 17A and B.

Example 13 Expression and Purification of huDS6 in CHO Cells andAffinity Measurements

To determine whether the humanized DS6 versions retained the bindingaffinity of muDS6 it was necessary to express and purify the antibodies.CHO cells were stably transfected with a chimeric version of DS6 (chDS6)having the human constant region and the murine variable region, or withhuDS6v1.01 or huDS6v1.21.

CHODG44 cells (4.32×10⁶ cells/plate) were seeded in 15 cm plates innon-selective media (Alpha MEM+nucleotides (Gibco), supplemented with 4mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% v/vFBS) and placed in a 37° C., 5% CO₂ humidified incubator. The followingday, the cells were transfected with the chDS6 expression plasmid usinga modified version of the Qiagen recommended protocol for PolyfectTransfection. The non-selective media was aspirated from the cells. Theplates were washed with 7 ml of pre-warmed (37° C.) PBS and replenishedwith 20 ml of non-selective media. The plasmid DNA (11 μg) was dilutedinto 800 μl of Hybridoma SFM (Gibco). Then, 70 μl of Polyfect (Qiagen)was added to the DNA/SFM mixture. The Polyfect mixture was then gentlyvortexed for several seconds and incubated for 10 min at ambienttemperature. Non-selective media (2.7 ml) was added to the mixture. Thisfinal mixture was incubated with the plated cells for 24 h.

The transfection mixture/media was removed from the plates and the cellswere then trypsinized and counted. The cells were then plated inselective media (Alpha MEM-nucleotides, supplemented with 4 mML-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 10% v/v FBS,1.25 mg/ml G418) in 96 well plates (250 μl/well) at various densities(1800, 600, 200, and 67 cells/well). The cells were incubated for 2-3weeks, supplementing media if necessary. Wells were screened forantibody production levels using a quantitative ELISA. An Immulon 2HB 96well plate was coated with goat anti-human IgG F(ab)₂ antibody (JacksonImmunoresearch; 1 μg/well in 100 μl 50 mM sodium carbonate buffer pH9.6) and incubated for 1.5 h at ambient temperature, with rocking. Allsubsequent steps were conducted at ambient temperature. The wells werewashed twice with T-TBS (0.1% Tween-20, TBS) and blocked with 200 μl ofblocking buffer (1% BSA, T-TBS) for 1 h. The wells were washed twicewith T-TBS. In a separate plate, the antibody standard, EM164 (100ng/ml), and culture supernatants were serially diluted (1:2 or 1:3) inblocking buffer. These dilutions (100 μl) were transferred to the ELISAplate and incubated for 1 h. The wells were washed 3 times with T-TBSand incubated with 100 μl of goat anti-human IgG Fc-AP (JacksonImmunoResearch) diluted 1:3000 in blocking buffer for 45 min. After 5washes with T-TBS, the wells were developed using 100 μl of PNPPdevelopment reagent (10 mg/ml PNPP (p-Nitrophenyl Phosphate, DisodiumSalt; Pierce), 0.1 M diethanolamine pH 10.3 buffer) for 25 min. Theabsorbance at 405 μm was measured in an ELISA plate reader. Absorbancereadings (of the culture supernatant) in the linear portion of thestandard curve were used to determine the antibody levels.

The highest producing clones, identified by the ELISA, were thensubcloned, expanded, and frozen cell stocks were prepared.

For expression of huDS6v1.01 and huDS6v1.21, DG44 CHO cells (Dr.Lawrence Chasin, Columbia University, NY) were cultured in Alpha MEMwith ribonucleosides and deoxyribonucleosides (Gibco catalog #12571,Grand Island, N.Y.). The medium was supplemented with 10% fetal bovineserum (HyClone catalog # SH30071.03, Logan, Utah), 1% gentamicin(Mediatech catalog# 30-005-CR, Herndon, Va.), and 2 mM L-glutamine(L-glut) (BioWhittaker catalog#17-605E, Walkersville, Md.). Thisformulation was termed CHO Complete Medium.

DG44 CHO cells (5×10⁶) were transfected with 50 μg of huDS6 plasmid DNA.Prior to transfection, cells were removed from flasks with trypsin(Gibco catalog # 15090-046, Grand Island, N.Y.) and washed two timeswith unsupplemented Alpha MEM lacking ribonucleosides anddeoxyribonucleosides (Gibco catalog #12561, Grand Island, N.Y.). Thiswas termed Wash Medium. Cells were mixed with plasmid DNA in 0.4 cm gapelectrode cuvettes (BioRad catalog # 1652088, Hercules, Pa.). They wereplaced on ice for two minutes, and then pulsed at 1,000 μF and 260 voltsin a BioRad electroporation apparatus. Following electroporation, cellswere incubated on ice for two minutes. The cells were then plated infive 24 well plates (Costar catalog # 3524) in CHO Complete Medium andwere maintained in a 37° C. incubator with 5% CO₂. After 48 hours, themedium was removed from the wells. Wells were rinsed once with WashMedium and fed with Alpha MEM without ribonucleosides anddeoxyribonucleosides (Gibco catalog #12561, Grand Island, N.Y.)supplemented with 1% gentamicin, 2 mM L-glut, 10% dialyzed fetal bovineserum (Gibco catalog # 26400-044, Grand Island, N.Y.), and 1.25 mg/mLgeneticin (G418) (Gibco catalog # 11811, Grand Island, N.Y.). Thiscomplete formulation was termed Selection Medium. Clones were incubatedin Selection Medium for approximately two weeks at which time they werescreened for antibody production by Quantitative ELISA. The highestproducing clones were then subcloned, expanded, and frozen cell stockswere prepared.

To produce sufficient amount of antibody to purify, cells were expandedonto 15 cm plates (˜1×10⁶ cells/plate) with 30 ml of selective mediasupplemented with Ultra Low IgG FBS (Gibco) and incubated for 1 week.Culture supernatants were collected into 250 ml conical tubes, spun downin a tabletop centrifuge (2000 rpm, 5 min, 4° C.), and thensterile-filtered through a 0.2 μm filter apparatus.

For purification of DS6, pellets of NaOH were added to the filteredculture supernatants to a final pH of 8.0. A Hi Trap rProtein A column(Amersham) was equilibrated with 20-50 column volumes of binding buffer.The supernatant was loaded onto the column using a peristaltic pump.Then, the column was washed with 50 column volumes of binding buffer.The bound antibody was eluted off of the column using elution buffer(100 mM acetic acid, 50 mM NaCl, pH 3) into tubes set in a fractioncollector. The eluted antibody was neutralized using neutralizationbuffer (2 M K₂HPO₄, pH 10.0) and dialyzed overnight in PBS. The dialyzedantibody was filtered through a 0.2 μm syringe filter. The absorbance at280 nm was measured to determine the final protein concentration.

The affinity of the purified huIgG was compared with muDS6 by flowcytometry. In the first set of experiments direct binding to aCA6-expressing cell line, KB, was measured. As shown in FIG. 18 themuDS6, chDS6, huDS6v1.01 and huDS6 v1.21 show very similar affinitieswith apparent Kds of 0.82 nM, 0.69 nM, 0.82 and 0.85 nM, respectively,suggesting that resurfacing has not disrupted the CDRs. To confirm thatthe huDS6 versions retain the affinity of muDS6, competitive bindingexperiments were conducted. The advantage of this format is that thesame detection system is used for both murine and human antibodies; thatis biotin-muDS6/streptavidin-DTAF. The results of the competitionbinding assay comparing the ability of muDS6, chDS6, huDS6v1.01 andhuDS6v1.21 to compete with biotin-DS6 is shown in FIG. 19. The apparentEC₅₀ are 1.9 nM, 1.7 nM, 3.0 and 1.9 nM for muDS6, chDS6, huDS6 v1.01,and huDS6 v1.21, respectively. These results indicate that resurfacingof muDS6 to produce a humanized DS6 causes little reduction in bindingaffinity.

Example 14 Preparation of muDS6-DM1 Cytotoxic Conjugate

The muDS6 antibody (8 mg/ml) was modified using 8-fold molar excess ofN-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP) to introducedithiopyridyl groups. The reaction was carried out in 95% v/v Buffer A(50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v/v DMA for 2 h atroom temperature. The slightly turgid reaction mixture was gel-filteredthrough a NAP or Sephadex G25 column (equilibrated in Buffer A). Thedegree of modification was determined by measuring the absorbance of theantibody at 280 nm and the DTT released 2-mercaptopyridine (Spy) at 280and 343 nm. Modified muDS6 was then conjugated at 2.5 mg Ab/mL using a1.7-fold molar excess ofN^(2′)-deacetyl-N-^(2′)(3-mercapto-1-oxopropyl)-maytansine (L-DM1) overSPy. The reaction was carried out in Buffer A (97% v/v) with DMA (3%v/v). The reaction was incubated at room temperature overnight for ˜20h. The opaque reaction mixture was centrifuged (1162×g, 10 min) and thesupernatant was then gel-filtered through a NAP-25 or S300 (Tandem 3,3×26/10 desalting columns, G25 medium) column equilibrated in Buffer B(1×PBS pH 6.5). The pellet was discarded. The conjugate wassterile-filtered using a 0.22 μm Millex-GV filter and was dialyzed inBuffer B with a Slide-A-Lyzer. The number of DM1 molecules linked permolecule of muDS6 was determined by measuring the absorbance at both 252nm and 280 nm of the filtered material. The DM1/Ab ratio was found to be4.36 and the step yield of conjugated MUDS6 was 55%. The conjugatedantibody concentration was 1.32 mg/mL. The purified conjugate wasbiochemically characterized by size exclusion chromography (SEC) andfound to be 92% monomer. Analysis of DM1 in the purified conjugatedindicated that 99% was covalently bound to antibody. In FIG. 20, flowcytometric binding of the muDS6-DM1 conjugate and unmodified muDS6 toCaov-3 cells shows that conjugation of muDS6 results in only a slightloss of affinity.

Example 15 In Vitro Cytotoxicity of muDS6-DM1

As a naked antibody, muDS6 has shown no proliferative or growthinhibitive activity in cell cultures (FIG. 21) However, when muDS6 isincubated with cells in the presence of a DM1 conjugate to Goatanti-mouse IgG heavy and light chain, muDS6 is very effective attargeting and delivering this conjugate to the cell resulting inindirect cytotoxicity (FIG. 21). To further test the inherent activityof naked muDS6, a complement-dependent cytotoxicity (CDC) assay usingmuDS6 was conducted. HPAC and ZR-75-1 cells (25000 cells/well) wereplated in 96 well plates, in the presence of 5% human or rabbit serumand various dilutions of muDS6, in 200 μl of RHBP media (RPMI-1640, 0.1%BSA, 20 mM HEPES (pH 7.2-7.4), 100 U/ml penicillin and 100 ug/mlstreptomycin). The cells were incubated for 2 h at 37° C. Then AlamarBlue (10% of final concentration) reagent (Biosource) was added to thesupernatant. The cells were incubated for 5-24 hrs before measuringfluorescence. Murine DS6 had no effect in a complement-dependentcytotoxicity (CDC) assay (FIG. 22) This suggests that the therapeuticapplication of muDS6 would require the conjugation of a toxic effectormolecule.

The cytotoxicity of maytansinoid conjugated muDS6 antibody was examinedusing 2 different assay formats in various DS6 positive cell lines.Clonogenic assays were conducted where cells (1000-2500 cells/well) wereplated on 6-well plates in 2 ml of conjugate diluted in culture media.The cells were continuously exposed to the conjugate at severalconcentrations, generally between 3×10⁻¹¹ M to 3×10⁻⁹ M, and wereincubated in a 37° C., 6% CO₂ humidified chamber for 5-9 days. The wellswere washed with PBS and the colonies were stained with a 1% w/v crystalviolet/10% v/v formaldehyde/PBS solution. Unbound stain was then washedthoroughly from the wells with distilled water, and the plates wereallowed to dry. The colonies were counted using a Leica StereoZoom 4dissecting microscope.

Plating efficiency (PE) was calculated as the number of colonies/numberof cells plated. Surviving fraction was calculated as PE of treatedcells/PE of non-treated cells. The IC₅₀ concentration was determined bygraphing the surviving fraction of cells vs. the molar concentration ofthe conjugate. In a clonogenic assay (FIG. 23), muDS6-DM1 was effectivein killing Caov-3 cells with an estimated IC₅₀ of 800 pM. Antigennegative cells, A375, were only slightly affected by the conjugate at aconcentration of 3×10⁻⁹ M, the highest concentration of muDS6-DM1tested, demonstrating that the cell killing activity of the conjugate isdirected specifically toward antigen-expressing cells. However, despiteapparent sensitivity to maytansine, many of the other DS6 positive celllines were not particularly sensitive to the immunoconjugate. All of thecervical cell lines (HeLa, KB, and WISH) were sensitive to the conjugatewhereas only a select number of the ovarian and breast cell lines showedany cytotoxic affects. None of the pancreatic cell lines appeared tohave been affected.

In the MTT assay, cells were seeded in 96-well plates at a density of1000-5000 cells/well. The cells were plated with serial dilutions ofeither naked muDS6 or muDS6-DM1 immunoconjugate in 200 μl of culturemedia. The samples were run in triplicate. The cells andantibody/conjugate mixtures were then incubated for 2-7 d, at which timecell viability was assessed by an MTT([3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay.MTT (50 μg/well) was added to the culture supernatant and allowed toincubate for 3-4 h at 37° C. The media was removed and the MTT formazanwas solubilized in DMSO (175 μl/well). The absorbance at 540-545 nm wasmeasured. In a MTT cell viability assay (FIG. 24C), the immunoconjugatewas able to effectively kill Caov-3 cells with an estimated IC₅₀ of 1.61nM. The wells with the highest concentrations of conjugate contained noviable cells as compared to naked antibody which had no effect (FIGS. 21and 24).

The results of the MTT assays on the other cell lines were slightlydifferent (FIGS. 24A, B, and D-I). In many cases, although somecytotoxicity was seen, the conjugate was unable to completely kill theentire cell population (with the exception of WISH cells). BT-20,OVCAR5, and HPAC cells were particularly resistant: in the highestconjugate concentration (32 nM) wells, over 50% of the cells were stillviable.

Example 16 In Vivo Conjugate Anti-Tumor Activity

To demonstrate the in vivo activity of the muDS6-DM1 conjugate, humantumor xenografts were established in SCID mice. A subcutaneous model ofthe human cervical carcinoma cell-line, KB, was developed. KB cells weregrown in vitro, collected, and 5×10⁶ cells in a 100 μL of serum freemedium were injected under the right shoulder of each mouse and allowedto grow for 6 days to an average tumor volume of 144±125 mm³ at whichtime drug treatment was initiated. Mice were given either PBS, conjugateat 150 μg/kg DM1, or conjugate at 225 μg/kg DM1 (2 mice per group)intravenously every day for 5 days. Toxic responses were monitored dailyduring the treatment. Tumor volumes (FIG. 25A) and corresponding bodyweights (FIG. 25B) were monitored throughout the study.

The KB tumors treated with PBS control grew rapidly with a doubling timeof about 4 days. In contrast, both groups of mice treated with conjugateexhibit complete tumor regression 14 days and 18 days after treatmentinitiation for the 225 μg/kg and 150 μg/kg dose groups, respectively. Atthe 150 μg/kg dose the tumor delay was approximately 70 days. Treatmentat 225 μg/kg resulted in cures as there was no evidence of tumorrecurrence at the termination of the study on day 120. As seen in FIG.25B the mice in the 150 μg/kg group showed no weight loss indicatingthat the dose was well tolerated. At the higher dose the mice experienceonly a temporary 3% reduction in body weight. During the 5-day treatmentcourse, mice exhibited no visible signs of toxicity. Taken together,this study demonstrates that muDS6-DM1 treatment can cure mice of KBxenograft tumors at a non-toxic dose.

muDS6-DM1 activity was further tested on a panel of subcutaneousxenograft models (see FIG. 26). The tumor cell-lines used to makexenografts displayed a range of in vitro maytansine sensitivities andCA6 epitope densities (Table 9 below). OVCAR5 cells and TOV-21G areovarian tumor cell lines; HPAC is a pancreatic tumor cell line; HeLa isa cervical tumor cell line. OVCAR5 and TOV-21G cells have low surfaceCA6 expression; HeLa cells have an intermediate level of surface CA6expression; HPAC cells have a high CA6 density of surface expression.TOV-21G and HPAC cells are maytansine sensitive; OVCAR5 and HeLa cellsare 2-7-fold less maytansine sensitive. TABLE 9 Clonogenic AssayClonogenic MTT Assay Cell Apparent Maytansine Assay Conjugate ConjugateLine MMF* Kd (M) IC₅₀ (M) IC₅₀ (M) EC₅₀ (M) BT-20 232.20 9.14 × 10⁻¹⁰3.50 × 10⁻¹⁰ >3.00 × 10⁻⁰⁹  1.44 × 10⁻⁰⁸ BT-483 1911.00 1.37 × 10⁻⁰⁸1.50 × 10⁻¹⁰ 1.00 × 10⁻¹⁰ N/A Caov-3 465.20 5.48 × 10⁻⁰⁹ 3.20 × 10⁻¹¹8.00 × 10⁻¹⁰ 1.61 × 10⁻⁰⁹ Caov-4 149.00 4.04 × 10⁻⁰⁹ 6.00 × 10⁻¹⁰ >3.00× 10⁻⁰⁹  N/A HeLa 242.50 6.94 × 10⁻¹⁰ 1.00 × 10⁻¹⁰ 1.80 × 10⁻⁰⁹ N/A HPAC2228.00 2.35 × 10⁻⁰⁸ 5.50 × 10⁻¹¹ 1.80 × 10⁻⁰⁹ 1.84 × 10⁻⁰⁹ HPAF-II266.50 2.81 × 10⁻⁰⁹ 6.00 × 10⁻¹⁰ >3.00 × 10⁻⁰⁹  1.00 × 10⁻⁰⁸ Hs766T182.90 2.32 × 10⁻⁰⁹ >3.00 × 10⁻⁰⁹  >3.00 × 10⁻⁰⁹  >3.20 × 10⁻⁰⁸  KB119.56 1.11 × 10⁻¹⁰ 3.00 × 10⁻¹¹ 1.40 × 10⁻⁰⁹ 3.01 × 10⁻⁰⁹ OVCAR5 97.101.47 × 10⁻⁰⁹ 3.20 × 10⁻¹⁰ >3.00 × 10⁻⁰⁹  8.46 × 10⁻⁰⁷ T-47D 559.58 3.42× 10⁻⁰⁹ 1.20 × 10⁻¹⁰ >3.00 × 10⁻⁰⁹  N/A TOV-21G 87.79 3.07 × 10⁻¹⁰ 4.80× 10⁻¹¹ 2.00 × 10⁻⁰⁹ 6.88 × 10⁻⁰⁹ WISH 1133.55 2.38 × 10⁻⁰⁹ 9.00 × 10⁻¹¹4.60 × 10⁻¹⁰ 6.69 × 10⁻¹⁰ ZR-75-1 811.67 4.30 × 10⁻⁰⁹ 1.00 × 10⁻¹⁰ N/A9.45 × 10⁻¹⁰*average maximum relative mean fluorescence

The 4 cell-lines were grown in vitro, collected, and 1×07 cells in a 100μL of serum free medium were injected under the right shoulder of eachmouse (6 mice per model) and allowed to grow for 6 days to an averagetumor volume of 57.6±6.7 and 90.2±13.4 mm³ for the test and controlgroups respectively of OVCAR5, 147.1±29.6 and 176.2±18.9 mm³ for thetest and control groups respectively of HPAC, 194.3±37.2 and 201.7±71.7mm³ for the test and control groups respectively of HeLa, and 96.6±22.8and 155.6±13.4 mm³ for the test and control groups respectively ofTOV-21G, at which time drug treatment was initiated. For each modelthree control mice were treated with two weekly doses of PBS and threetest mice were treated with two weekly doses of conjugate (600 μg/kgDM1) intravenously. Toxic responses were monitored daily during thetreatment and tumor volumes and body weights were monitored throughoutthe study. The conjugate efficacy for the various models is showngraphically in FIGS. 26A, C, E, and G and the corresponding body weightsare plotted in FIGS. 26B, D, F, and H. OVCAR5, TOV-21G, and HPACcell-lines form aggressive tumors as can be seen in the PBS controls foreach model. The HeLa model had about a 3 week lag period beforebeginning exponential growth. In all models, DS6-DM1 conjugate treatmentresulted in a complete tumor regression in all mice. For the TOV-21G,HPAC, and HeLa models the mice remain tumor-free at day 61. In theOVCAR5 model tumors recurred at about day 45 after tumor inoculation.Thus, muDS6-DM1 treatment in this model results in a tumor growth delayof approximately 34 days. The growth delay is significant as OVCAR5cells are less maytansine-sensitive and have low CA6 epitope expression.In models where either the CA6 epitope density is higher or the modelhas greater maytansine sensitivity, the tumor regression is more robust.It is important to note that only 2 doses were administered. Clearly thedosing schedule used in this study was not toxic to the mice as noweight loss was observed. It is likely that cures could be achieved withadditional or higher conjugate doses.

Human ovarian cancer is largely a disease of the peritoneum. OVCAR5cells grow aggressively as an intraperitoneal (IP) model in SCID miceforming tumor nodules and producing ascites in a manner similar to humandisease. To demonstrate activity in an IP model, muDS6-DM1 was used totreat mice bearing OVCAR5 IP tumors (FIG. 27). OVCAR5 cells were grownin vitro, harvested and 1×10⁷ cells in 100 μL of serum free medium wereinjected intraperitoneally. Tumors were allowed to grow for 6 days atwhich time treatment was initiated. Mice were treated weekly for 2 weekswith either PBS or DS6-DM1 conjugate at a dose of 600 μg/kg DM1 andmonitored for weight loss resulting from peritoneal disease. By day 28,the PBS group of mice had lost greater than 20% body weight and wereeuthanized. The treated group was sacrificed at day 45 after exceeding20% body weight loss. This study demonstrates that muDS6-DM1 is able todelay tumor growth in the aggressive OVCAR5 IP model despite the factthat OVCAR5 cells are less sensitive to maytansine and have few CA6epitopes per cell. Because the dosing schedule used elicited no visiblesigns of toxicity, it is likely that additional and higher doses couldbe used to achieve further tumor growth delay or cures.

Example 17 Synthesis and Characterization of DS6-SPP-MM1-202 TaxoidCytotoxic Conjugate

muDS6 was modified with the N-sulfosuccinimidyl4-nitro-2-pyridyl-pentanoate (SSNPP) linker. To 50 mg of muDS6 Ab in 90%Buffer A, 10% DMA was added 10 equivalents of SSNPP in DMA. The finalconcentration of Ab was 8 mg/ml. The reaction was stirred for 4 hours atroom temperature, then purified by G25 chromatography. The extent ofantibody modification was measured spectophotometrically using theabsorbance at 280 nm (antibody) and 325 (linker) and found to have 3.82linkers/antibody. Recovery of the antibody was 43.3 mg giving an 87%yield. Conjugation of muDS6-nitroSPP was conjugated with Taxoid MM1-202(1812 P.16). Conjugation was carried out on a 42 mg scale in 90% BufferA, 10% DM1. The taxoid was added in 4 aliquots of 0.43 eq/Linker (eachaliquot) over a period of about 20 hours. By this time the reaction hadturned noticeably cloudy. After G25 purification the resultingconjugate, recovered in about 64% yield had about 4.3 taxoids/Ab andabout 1 equivalent of unreacted linker left. To quench unreacted linker,1 equivalent of cysteine/unreacted linker was added to the conjugatewith stirring overnight. A definite yellowish tinge was noticeable uponcysteine addition indicating release of thiopyridine. The reactionsolution was then dialyzed in Buffer B/0.01% Tween 20 followed byfurther dialysis in Buffer B alone over several days. The finalconjugate had 2.86 drugs/antibody. The antibody recovery was 14.7 mg,giving a 35% yield overall. Conjugate was further biochemicallycharacterized by SEC and found to have 89% monomer, 10.5% dimer and 0.5%higher molecular weight aggregate.

The results of a flow cytometry analysis comparing the binding ofmuDS6-SPP-MM1-202 taxoid versus muDS6 antibody on HeLa cells is shown inFIG. 28. The results indicate that muDS6 retains binding activity whenconjugated to a taxane.

Example 18 In Vitro and In Vivo Activity of Humanized DS6 Conjugate

A huDS6v1.01-SPDB-DM4 conjugate was constructed. This conjugate issimilar to the muDS6-SPP-DM1 conjugate described in Example 14 exceptthat the linker/maytansine drug part of the conjugate differs in thestructure around the disulfide bond; the muDS6-SPP-DM1 conjugate has onemethyl group hindrance on the disulfide carbon on the antibody side ofthe linker while the SPDB-DM4 conjugate has two methyl group hindranceon the disulfide carbon on the maytansine side of the linker.

The huDS6v1.01 antibody (8 mg/ml) was modified using 8-fold molar excessof N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) to introducedithiopyridyl groups. The reaction was carried out in 95% v/v Buffer A(50 mM KPi, 50 mM NaCl, 2 mM EDTA, pH 6.5) and 5% v/v ethanol for 1.5 hat room temperature. The reaction mixture was gel-filtered through a 15ml Sephadex G25 column (equilibrated in Buffer A). The degree ofmodification was determined by measuring the absorbance of the antibodyat 280 nm and the DTT released 2-mercaptopyridine (Spy) at 280 and 343nm. Modified DS6 was then conjugated at 1.8 mg Ab/mL using a 1.7-foldmolar excess ofN^(2′)-deacetyl-N-^(2′)(4-methyl-4-mercapto-1-oxopentyl)-maytansine(L-DM4) over SPy. The reaction was carried out in Buffer A (97% v/v)with DMA (3% v/v). The reaction was incubated at room temperatureovernight for ˜20 h. In contrast to the conjugation of muDS6, thereaction mixture was clear and immediately underwent gel-filtrationthrough a NAP 15 ml G25 column equilibrated in Citrate buffer (20 mMcitrate, 135 mM NaCl, pH 5.5). The conjugate was sterile-filtered usinga 0.22 μm Millex-GV filter. The number of DM4 molecules linked permolecule of DS6 was determined by measuring the absorbance at both 252nm and 280 nm of the filtered material. The DM4/Ab ratio was found to be3.2 and the step yield of conjugated DS6 was 69%. The conjugatedantibody concentration was 1.51 mg/mL. The purified conjugate wasbiochemically characterized by size exclusion chromotography (SEC) andfound to be 92.5% monomer. Analysis of DM4 in the purified conjugatedindicated that >99% was covalently bound to antibody.

In FIG. 29A, flow cytometric binding of the huDS6v1.01-DM4 conjugate andunmodified DS6 to KB cells shows that conjugation of huDS6v1.01 resultsin essentially no loss of affinity. The binding was conductedessentially as described for FIG. 20 except that KB cells were usedrather than CaOv-3 cells. In vitro cytotoxicity of huDS6v1.01 was testedessentially as described in FIG. 24G. huDS6v1.01 killed WISH cells withan IC₅₀ of 4.4×10⁻¹⁰ M whereas unconjugated huDS6v1.01 showed nocytotoxic activity.

The in vivo activity of huDS6v1.01-DM4 was tested on the HPAC pancreaticmodel. HPAC cells were inoculated on day 0, and immunoconjugatetreatments were given on day 13. PBS control animals were euthanizedonce tumor volumes exceeded 1000 mm³. The conjugate was given at a doseof either 200 μg/kg or 600 μg/kg DM4, corresponding to an antibodyconcentration 15 mg/kg and 45 mg/kg, respectively. Tumor volume (FIG.30A) and body weight (FIG. 30B) of the mice were monitored during thecourse of the study. The huDS6v1.01-DM4 showed potent anti-tumoractivity at 200 μg/kg DM4 with all mice achieving complete tumorregression. The control B4-DM4 conjugate recognizing an antigen notexpressed on HPAC xenografts had essentially no activity at 200 μg/kg.The lack of body weight loss (FIG. 30B) of the mice indicates that thetreatment with 200 μg/kg conjugate is below the maximum tolerated dose.This result demonstrates that a humanized version of DS6 is able tomediate targeted delivery of a maytansinoid drug resulting in potentanti-tumor activity.

1. An antibody or epitope-binding fragment thereof comprising at least one heavy chain variable region or fragment thereof and at least one light chain variable region or fragment thereof, wherein said heavy chain variable region or fragment thereof has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11: (SEQ ID NO: 9) QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDS VPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.


2. The antibody or epitope-binding fragment thereof of claim 1, wherein said heavy chain variable region or fragment thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.
 3. The antibody or epitope-binding fragment thereof of claim 1, wherein said heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.
 4. The antibody or epitope-binding fragment thereof of claim 1, wherein said light chain variable region or fragment thereof has at least 90% sequence identity to an amino acid sequence represented by SEQ ID NO:7 or SEQ ID NO:8: (SEQ ID NO: 7) QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYST SSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAG TKLELKR (SEQ ID NO: 8) EVILTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYST SSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAG TKLELKR.


5. The antibody or epitope-binding fragment thereof of claim 1, wherein said light chain variable region or fragment thereof has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
 6. The antibody or epitope-binding fragment thereof of claim 1, wherein said light chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
 7. The antibody or epitope-binding fragment thereof of claim 1, wherein said heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and wherein said light chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8.
 8. A polynucleotide encoding an antibody or an epitope-binding fragment thereof according to claim
 1. 9. A polynucleotide encoding an antibody or an epitope-binding fragment thereof according to claim
 4. 10. A polynucleotide encoding an antibody or an epitope-binding fragment thereof according to claim
 7. 11. A polynucleotide encoding a heavy chain of an antibody or an epitope-binding fragment thereof according to claim
 1. 12. An expression vector comprising the polynucleotide of claim
 8. 13. An expression vector comprising the polynucleotide of claim
 9. 14. An expression vector comprising the polynucleotide of claim
 10. 15. A host cell comprising an expression vector of claim
 12. 16. A host cell comprising an expression vector of claim
 13. 17. A host cell comprising an expression vector of claim
 14. 18. A method of preparing an antibody or an epitope-binding fragment thereof comprising culturing the host cell of claim 15 under conditions promoting expression of said antibody or an epitope-binding fragment thereof and recovering said polypeptide from the cell culture, wherein said antibody or an epitope-binding fragment thereof comprises at least one heavy chain variable region or fragment thereof and at least one light chain variable region or fragment thereof, wherein said heavy chain variable region or fragment thereof has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11: (SEQ ID NO: 9) QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDS VPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA.


19. A method of preparing an antibody or an epitope-binding fragment thereof comprising culturing the host cell of claim 16 under conditions promoting expression of said antibody or an epitope-binding fragment thereof and recovering said polypeptide from the cell culture, wherein said heavy chain variable region or fragment thereof has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11: (SEQ ID NO: 9) QAYLQQSGAELVRSGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKKGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGDS VPFAYWGQGTLVTVSA (SEQ ID NO: 10) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKFQGKATLTADTSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA (SEQ ID NO: 11) QAQLVQSGAEVVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGY IYPGNGATNYNQKFQGKATLTADPSSSTAYMQISSLTSEDSAVYFCARGD SVPFAYWGQGTLVTVSA, and

wherein said light chain variable region or fragment thereof has at least 90% sequence identity to an amino acid sequence represented by SEQ ID NO:7 or SEQ ID NO:8: (SEQ ID NO:7) QIVLTQSPAIMSASPGEKVTITCSAHSSVSFMHWFQQKPGTSPKLWIYST SSLASGVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSSFPLTFGAG TKLELKR (SEQ ID NO:8) EIVLTQSPATMSASPGERVTITCSAHSSVSFMHWFQQKPGTSPKLWIYST SSLASGVPARFGGSGSGTSYSLTISSMEAEDAATYYCQQRSSFPLTFGAG TKLELKR.


20. A method of preparing an antibody or an epitope-binding fragment thereof comprising culturing the host cell of claim 17 under conditions promoting expression of said antibody or an epitope-binding fragment thereof and recovering said polypeptide from the cell culture, wherein said heavy chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and wherein said light chain variable region or fragment thereof has the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:8. 